Materials and Methods for Treatment of Duchenne Muscular Dystrophy

ABSTRACT

The present application provides materials and methods for treating a patient with Duchenne Muscular Dystrophy (DMD) both ex vivo and in vivo. In addition, the present application provides materials and methods for editing a dystrophin gene in a cell by genome editing.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/763,328filed on Jun. 22, 2018, which is a 371 application of PCT/IB2016/001679filed Oct. 28, 2016, which claims the benefit of U.S. ProvisionalApplication No. 62/247,484 filed Oct. 28, 2015 and U.S. ProvisionalApplication No. 62/324,064 filed Apr. 18, 2016, all of which areincorporated herein in their entirety by references.

TECHNICAL FIELD

The present application provides materials and methods for treating apatient with Duchenne Muscular Dystrophy (DMD), both ex vivo and invivo. In addition, the present application provides materials andmethods for editing a dystrophin gene in a cell by genome editing.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form(filename: 160101PCT sequence listing_ST25: 286,928,896 bytes—ASCII textfile; created Oct. 28, 2016), which is incorporated herein by referencein its entirety and forms part of the disclosure.

BACKGROUND

Duchenne Muscular Dystrophy (DMD) is a severe X-linked recessiveneuromuscular disorder effecting approximately 1 in 4,000 live malebirths. Patients are generally diagnosed by the age of 4, and wheelchair bound by the age of 10. Most patients do not live past the age of25 due to cardiac and/or respiratory failure. Existing treatments arepalliative at best. The most common treatment for DMD is steroids, whichare used to slow the loss of muscle strength. However, because most DMDpatients start receiving steroids early in life, the treatment delayspuberty and further contributes to the patient's diminished quality oflife.

DMD is caused by mutations in the dystrophin gene (Chromosome X:31,117,228-33,344,609 (Genome Reference Consortium—GRCh38/hg38)). With agenomic region of over 2.2 megabases in length, dystrophin is the secondlargest human gene. The dystrophin gene contains 79 exons that areprocessed into an 11,000 base pair mRNA that is translated into a 427kDa protein. Functionally, dystrophin acts as a linker between the actinfilaments and the extracellular matrix within muscle fibers. TheN-terminus of dystrophin is an actin-binding domain, while theC-terminus interacts with a transmembrane scaffold that anchors themuscle fiber to the extracellular matrix. Upon muscle contraction,dystrophin provides structural support that allows the muscle tissue towithstand mechanical force. DMD is caused by a wide variety of mutationswithin the dystrophin gene that result in premature stop codons andtherefore a truncated dystrophin protein. Truncated dystrophin proteinsdo not contain the C-terminus, and therefore cannot provide thestructural support necessary to withstand the stress of musclecontraction. As a result, the muscle fibers pull themselves apart, whichleads to muscle wasting.

Becker Muscular Dystrophy (BMD) is a less severe form of musculardystrophy compared to DMD. While BMD is also caused by mutations withinthe dystrophin gene, BMD mutations maintain the dystrophin readingframe. BMD dystrophin proteins contain internal deletions, but alsoretain portions of both the N and C termini. Therefore, the BMDdystrophin protein is shorter than the wild type protein, but can stillfunction as a linker between the actin filaments and the extracellularmatrix. In fact, depending on the size of the internal deletion, BMDpatients may have only minor symptoms. As a result, most researchefforts have been focused on converting the severe DMD phenotype to aless severe BMD phenotype.

Genome engineering refers to the strategies and techniques for thetargeted, specific modification of the genetic information (genome) ofliving organisms. Genome engineering is a very active field of researchbecause of the wide range of possible applications, particularly in theareas of human health; the correction of a gene carrying a harmfulmutation, for example, to explore the function of a gene. Earlytechnologies developed to insert a gene into a living cell, such astransgenesis, were often limited by the random nature of the insertionof the new sequence into the genome. The new gene was usually positionedblindly, and may have inactivated or disturbed the functioning of othergenes, or even caused severe unwanted effects. Furthermore, thesetechnologies generally offered no degree of reproducibility, as therewas no guarantee that the new sequence would be inserted at the sameplace in two different cells. More recent genome engineering strategies,such as ZFNs, TALENs, HEs and MegaTALs, enable a specific area of theDNA to be modified, thereby increasing the precision of the correctionor insertion compared to early technologies, and offering some degree ofreproducibility. Despite this, such recent genome engineering strategieshave limitations.

Multiple studies suggest that genome engineering would be an attractivestrategy for treating DMD. One of the earliest approaches involvedengineering a mini-dystrophin gene that is less than 4 kb and can bepackaged into an adeno-associated virus (AAV) vector. This is areplacement gene therapy that has been tested experimentally in mouse(Wang, B., J. Li, and X. Xiao, Proc Natl Acad Sci USA, 2000. 97(25): p.13714-9) (Watchko, J., et al., Hum Gene Ther, 2002. 13(12): p. 1451-60)and dog models (Wang, Z., et al., Mol Ther, 2012. 20(8): p. 1501-7), anda phase I clinical trial suggested that there are problems associatedwith an immune response to the non-self synthetic epitopes (Mendell, J.R., et al., N Engl J Med, 2010. 363(15): p. 1429-37).

More recently, oligo-mediated exon skipping was used to restore thereading frame in the cells of DMD patients. In this strategy, shortoligos block splicing signals found in pre-mRNA and facilitate skippingof a single exon. Skipping of a single exon allows the transcriptionalmachinery to bypass the premature stop codon and produce a protein withintact N and C termini. Phase I/II clinical trials have shown thatweekly injections of anti-sense oligos induce exon skipping anddystrophin positive fibers (Cirak, S., et al., Lancet, 2011. 378(9791):p. 595-605). However, the major limitation of this type of treatment isthat it requires repeat dosing over the lifetime of the patient becausethe drug targets the pre-mRNA rather than the genomic locus. OngoingPhase II/111 clinical trials are evaluating delivery of exon skippingoligos via AAV for sustained expression, as well as delivery of multipleanti-sense oligos for facilitating multi-exon skipping strategies.

Despite efforts from researchers and medical professionals worldwide whohave been trying to address DMD, and despite the promise of genomeengineering approaches, there still remains a critical need fordeveloping safe and effective treatments for DMD, which is among themost prevalent and debilitating genetic disorders.

SUMMARY

The present disclosure presents an approach to address the genetic basisof DMD. By using genome engineering tools to create permanent changes tothe genome that can restore the dystrophin reading frame and restore thedystrophin protein activity with as few as a single treatment, theresulting therapy can correct the underlying genetic defect causing thedisease.

Provided herein are cellular, ex vivo and in vivo methods for creatingpermanent changes to the genome by deleting, inserting, or replacing(deleting and inserting) one or more exons or aberrant intronic spliceacceptor or donor sites in the dystrophin gene by genome editing andrestoring the dystrophin reading frame and restoring the dystrophinprotein activity, which can be used to treat Duchenne Muscular Dystrophy(DMD). Also provided herein are components, kits and compositions forperforming such methods. Also, provided are cells produced by suchmethods.

Provided herein is a method for editing a dystrophin gene in a humancell by genome editing, the method comprising the step of introducinginto the human cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the dystrophin gene thatresults in a permanent deletion, insertion, or replacement of one ormore exons or aberrant intronic splice acceptor or donor sites within ornear the dystrophin gene and results in restoration of the dystrophinreading frame and restoration of the dystrophin protein activity. Thehuman cell can be a muscle cell or muscle precursor cell.

Also provided herein is an ex vivo method for treating a patient (e.g.,a human) with Duchenne Muscular Dystrophy (DMD), the method comprisingthe steps of: i) creating a DMD patient specific induced pluripotentstem cell (iPSC); ii) editing within or near a dystrophin gene of theiPSC; iii) differentiating the genome-edited iPSC into a Pax7+ muscleprogenitor cell; and iv) implanting the Pax7+ muscle progenitor cellinto the patient.

The step of creating a patient specific induced pluripotent stem cell(iPSC) can comprise: a) isolating a somatic cell from the patient; andb) introducing a set of pluripotency-associated genes into the somaticcell to induce the somatic cell to become a pluripotent stem cell. Thesomatic cell can be a fibroblast. The set of pluripotency-associatedgenes is one or more of the genes selected from the group consisting ofOCT4, SOX2, KLF4, Lin28, NANOG and cMYC.

The step of editing within or near a dystrophin gene of the iPSC cancomprise introducing into the iPSC one or more deoxyribonucleic acid(DNA) endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the dystrophin gene thatresults in a permanent deletion, insertion, or replacement of one ormore exons or aberrant intronic splice acceptor or donor sites within ornear the dystrophin gene and results in restoration of the dystrophinreading frame and restoration of the dystrophin protein activity.

The step of differentiating the genome-edited iPSC into a Pax7+ muscleprogenitor cell can comprise contacting the genome-edited iPSC withspecific media formulations, including small molecule drugs; transgeneoverexpression; or serum withdrawal.

The step of implanting the Pax7+ muscle progenitor cell into the patientcan comprise implanting the Pax7+ muscle progenitor cell into thepatient by local injection into the desired muscle.

Also provided herein is an in vivo method for treating a patient (e.g.,a human) with Duchenne Muscular Dystrophy (DMD), the method comprisingthe step of editing a dystrophin gene in a cell of the patient. The cellcan be a muscle cell or muscle precursor cell.

The step of editing a dystrophin in a cell of the patient can compriseintroducing into the cell of the patient one or more deoxyribonucleicacid (DNA) endonucleases to effect one or more single-strand breaks(SSBs) or double-strand breaks (DSBs) within or near the dystrophin genethat results in a permanent deletion, insertion, or replacement of oneor more exons or aberrant intronic splice acceptor or donor sites withinor near the dystrophin gene and results in restoration of the dystrophinreading frame and restoration of the dystrophin protein activity.

The one or more DNA endonucleases can be a Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3,Csf4, or Cpf1 endonuclease; a homolog thereof, a recombinant of thenaturally occurring molecule thereof, a codon-optimized thereof,modified version thereof, and combinations of any of the foregoing.

The method can comprise introducing into the cell one or morepolynucleotides encoding the one or more DNA endonucleases. The methodcan comprise introducing into the cell one or more ribonucleic acids(RNAs) encoding the one or more DNA endonucleases. The one or morepolynucleotides or one or more RNAs can be one or more modifiedpolynucleotides or one or more modified RNAs. The one or more DNAendonuclease can be one or more proteins or polypeptides.

The method can further comprise introducing into the cell one or moreguide ribonucleic acids (gRNAs). The one or more gRNAs aresingle-molecule guide RNA (sgRNAs). The one or more gRNAs or one or moresgRNAs is one or more modified gRNAs or one or more modified sgRNAs. Theone or more DNA endonucleases can be pre-complexed with one or moregRNAs or one or more sgRNAs.

The method can further comprise introducing into the cell apolynucleotide donor template comprising at least a portion of thewild-type dystrophin gene or cDNA. The at least a portion of thewild-type dystrophin gene or cDNA can include at least a part of exon 1,exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10,exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18,exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26,exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34,exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42,exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50,exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58,exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66,exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74,exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions, syntheticintronic regions, fragments, combinations thereof, or the entiredystrophin gene or cDNA. The at least a portion of the wild-typedystrophin gene or cDNA can include exon 1, exon 2, exon 3, exon 4, exon5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13,exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21,exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29,exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37,exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45,exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53,exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61,exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69,exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77,exon 78, exon 79, intronic regions, synthetic intronic regions,fragments, combinations thereof, or the entire dystrophin gene or cDNA.The donor template can be a single or double stranded polynucleotide.

The method can further comprise introducing into the cell one or moreguide ribonucleic acid (gRNAs). The one or more DNA endonucleases can beone or more Cas9 or Cpf1 endonucleases that effect a pair ofsingle-strand breaks (SSBs) or double-strand breaks (DSBs), the firstSSB or DSB break at a 5′ locus and the second SSB or DSB break at a 3′locus, that results in a permanent deletion or replacement of one ormore exons or aberrant intronic splice acceptor or donor sites betweenthe 5′ locus and the 3′ locus within or near the dystrophin gene andresults in restoration of the dystrophin reading frame and restorationof the dystrophin protein activity. One gRNA can create a pair of SSBsor DSBs. One gRNA can comprise a spacer sequence that is complementaryto either the 5′ locus, the 3′ locus, or a segment between the 5′ locusand 3′ locus. A first gRNA can comprise a spacer sequence that iscomplementary to a segment of the 5′ locus and the second gRNA cancomprise a spacer sequence that is complementary to a segment of the 3′locus.

The one or more gRNAs can be one or more single-molecule guide RNAs(sgRNAs). The one or more gRNAs or one or more sgRNAs can be one or moremodified gRNAs or one or more modified sgRNAs. The one or more DNAendonucleases can be pre-complexed with the one or more gRNAs or one ormore sgRNAs.

There can be a deletion of the chromosomal DNA between the 5′ locus andthe 3′ locus.

The deletion can be a single exon deletion. The single exon deletion canbe a deletion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46,exon 50, exon 51, exon 52, or exon 53. The 5′ locus can be proximal to a5′ boundary of a single exon selected from the group consisting of exon2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon52, and exon 53. The 3′ locus can be proximal to a 3′ boundary of asingle exon selected from the group consisting of exon 2, exon 8, exon43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53.The 5′ locus can be proximal to a 5′ boundary and the 3′ locus can beproximal to the 3′ boundary of a single exon selected from the groupconsisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon50, exon 51, exon 52, and exon 53. Proximal to the boundary of the exoncan include the surrounding splice donors and acceptors of theneighboring intron.

The deletion can be a multi-exon deletion. The multi-exon deletion canbe a deletion of exons 45-53 or exons 45-55. The 5′ locus can beproximal to a 5′ boundary of multiple exons selected from the groupconsisting of exons 45-53 and exons 45-55. The 3′ locus can be proximalto a 3′ boundary of multiple exons selected from the group consisting ofexons 45-53 and exons 45-55. The 5′ locus can be proximal to a 5′boundary and a 3′ locus can be proximal to the 3′ boundary of multipleexons selected from the group consisting of exons 45-53 and exons 45-55.Proximal to the boundary of the exon can include the surrounding splicedonors and acceptors of the neighboring intron.

There can be a replacement of the chromosomal DNA between the 5′ locusand the 3′ locus. The replacement can be a single exon replacement. Thesingle exon replacement can be a replacement of exon 2, exon 8, exon 43,exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon70. The 5′ locus can be proximal to a 5′ boundary of a single exonselected from the group consisting of exon 2, exon 8, exon 43, exon 44,exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 3′locus can be proximal to a 3′ boundary of a single exon selected fromthe group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 5′ locus canproximal to a 5′ boundary and a 3′ locus can be proximal to the 3′boundary of a single exon selected from the group consisting of exon 2,exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52,exon 53, or exon 70. Proximal to the boundary of the exon can includethe surrounding splice donors and acceptors of the neighboring intron orneighboring exon.

The replacement can be a multi-exon replacement. The multi-exonreplacement can be a replacement of exons 45-53 or exons 45-55. The 5′locus can be proximal to a 5′ boundary of multiple exons selected fromthe group consisting of exons 45-53 or exons 45-55. The 3′ locus can beproximal to a 3′ boundary of multiple exons selected from the groupconsisting of exons 45-53 or exons 45-55. The 5′ locus can proximal to a5′ boundary and a 3′ locus can be proximal to the 3′ boundary ofmultiple exons selected from the group consisting of exons 45-53 orexons 45-55. Proximal to the boundary of the exon can include thesurrounding splice donors and acceptors of the neighboring intron orneighboring exon.

The method can further comprise introducing into the cell apolynucleotide donor template comprising at least a portion of the wildtype dystrophin gene or cDNA and the replacement is by homology directedrepair (HDR).

The at least a portion of the wild-type dystrophin gene or cDNA caninclude at least a part of exon 1, exon 2, exon 3, exon 4, exon 5, exon6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14,exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22,exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30,exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38,exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46,exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54,exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62,exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70,exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78,exon 79, intronic regions, synthetic intronic regions, fragments,combinations thereof, or the entire dystrophin gene or cDNA. The atleast a portion of the wild-type dystrophin gene or cDNA can includeexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9,exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17,exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25,exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33,exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41,exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49,exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57,exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65,exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73,exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions,synthetic intronic regions, fragments, combinations thereof, or theentire dystrophin gene or cDNA.

The method can further comprise introducing into the cell one guideribonucleic acid (gRNA) and a polynucleotide donor template comprisingat least a portion of the wild-type dystrophin gene. The one or more DNAendonucleases can be one or more Cas9 or Cpf1 endonucleases that effectone single-strand break (SSB) or double-strand break (DSB) at a locuswithin or near the dystrophin gene that facilitates insertion of a newsequence from the polynucleotide donor template into the chromosomal DNAat the locus that results in permanent insertion or correction of one ormore exons or aberrant intronic splice acceptor or donor sites within ornear the dystrophin gene and results in restoration of the dystrophinreading frame and restoration of the dystrophin protein activity. ThegRNA can comprise a spacer sequence that is complementary to a segmentof the locus.

The method can further comprise introducing into the cell one or moreguide ribonucleic acid (gRNAs) and a polynucleotide donor templatecomprising at least a portion of the wild-type dystrophin gene. The oneor more DNA endonucleases can be one or more Cas9 or Cpf1 endonucleasesthat effect a pair of single-strand breaks (SSBs) or double-strandbreaks (DSBs), the first at a 5′ locus and the second at a 3′ locus,within or near the dystrophin gene that facilitates insertion of a newsequence from the polynucleotide donor template into the chromosomal DNAbetween the 5′ locus and the 3′ locus that results in a permanentinsertion or correction of one or more exons or aberrant intronic spliceacceptor or donor sites between the 5′ locus and the 3′ locus within ornear the dystrophin gene and results in restoration of the dystrophinreading frame and restoration of the dystrophin protein activity.

One gRNA can create a pair of SSBs or DSBs. One gRNA can comprise aspacer sequence that is complementary to either the 5′ locus, the 3′locus, or a segment between the 5′ locus and the 3′ locus. A first gRNAcan comprise a spacer sequence that is complementary to a segment of the5′ locus and the second gRNA can comprise a spacer sequence that iscomplementary to a segment of the 3′ locus.

The one or more gRNAs can be one or more single-molecule guide RNAs(sgRNAs). The one or more gRNAs or one or more sgRNAs can be one or moremodified gRNAs or one or more modified sgRNAs. The one or more DNAendonucleases can be pre-complexed with the one or more gRNAs or one ormore sgRNAs.

There can be an insertion between the 5′ locus and the 3′ locus.

The insertion can be a single exon insertion. The single exon insertioncan be an insertion of exon 2, exon 8, exon 43, exon 44, exon 45, exon46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 5′ locus or 3′locus can be proximal to a boundary of a single exon selected from thegroup consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46,exon 50, exon 51, exon 52, exon 53, and exon 70. Proximal to theboundary of the exon can include the surrounding splice donors andacceptors of the neighboring intron or neighboring exon.

The insertion can be a multi-exon insertion. The multi-exon insertioncan be an insertion of exons 45-53 or exons 45-55. The 5′ locus or 3′locus can be proximal to a boundary of multiple exons selected from thegroup consisting of exons 45-53 or exons 45-55. Proximal to the boundaryof the exon can include the surrounding splice donors and acceptors ofthe neighboring intro.

The at least a portion of the wild-type dystrophin gene or cDNA caninclude at least a part of exon 1, exon 2, exon 3, exon 4, exon 5, exon6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14,exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22,exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30,exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38,exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46,exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54,exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62,exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70,exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78,exon 79, intronic regions, synthetic intronic regions, fragments,combinations thereof, or the entire dystrophin gene or cDNA. The atleast a portion of the wild-type dystrophin gene or cDNA can includeexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9,exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17,exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25,exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33,exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41,exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49,exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57,exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65,exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73,exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions,synthetic intronic regions, fragments, combinations thereof, or theentire dystrophin gene or cDNA.

The insertion or correction can be by homology directed repair (HDR).

The donor template can be a single or double stranded polynucleotide.

The Cas9 or Cpf1 mRNA, gRNA, and donor template can be each formulatedinto separate lipid nanoparticles or all co-formulated into a lipidnanoparticle.

The Cas9 or Cpf1 mRNA can be formulated into a lipid nanoparticle, andboth the gRNA and donor template can be delivered to the cell by anadeno-associated virus (AAV) vector.

The Cas9 or Cpf1 mRNA can be formulated into a lipid nanoparticle, andthe gRNA can be delivered to the cell by electroporation and donortemplate can be delivered to the cell by an adeno-associated virus (AAV)vector.

The dystrophin gene can be located on Chromosome X:31,117,228-33,344,609 (Genome Reference Consortium—GRCh38/hg38).

Also provided herein is one or more guide ribonucleic acids (gRNAs) forediting a dystrophin gene in a cell from a patient with DMD. The one ormore gRNAs and/or sgRNAs can comprise a spacer sequence selected fromthe group consisting of the nucleic acid sequences in SEQ ID Nos:1-1,410,472 of the Sequence Listing. The one or more gRNAs can be one ormore single-molecule guide RNAs (sgRNAs). The one or more gRNAs or oneor more sgRNAs can be one or more modified gRNAs or one or more modifiedsgRNAs.

Provided herein are cells that have been modified by the precedingmethods to permanently delete or correct one or more exons or aberrantintronic splice acceptor or donor sites within the dystrophin gene andrestore the dystrophin reading frame and restore the dystrophin proteinactivity. Further provided herein are methods for ameliorating DMD bythe administration of cells that have been modified by the precedingmethods to a DMD patient.

It is understood that the inventions described in this specification arenot limited to the examples summarized in this Summary. Various otheraspects are described and exemplified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of materials and methods for treatment of DMD disclosedand described in this specification can be better understood byreference to the accompanying figures, in which:

FIG. 1A is a plasmid (CTx-1) comprising a codon optimized gene for S.pyogenes Cas9 endonuclease. The CTx-1 plasmid also comprises a gRNAscaffold sequence, which includes a 20 bp spacer sequence from thesequences listed in SEQ ID NOs: 1-467,030 of the Sequence Listing or a19 bp spacer sequence from the sequences listed in SEQ ID NOs:1,410,430-1,410,472 of the Sequence Listing;

FIG. 1B is a plasmid (CTx-2) comprising a different codon optimized genefor S. pyogenes Cas9 endonuclease. The CTx-2 plasmid also comprises agRNA scaffold sequence, which includes a 20 bp spacer sequence from thesequences listed in SEQ ID NOs: 1-467,030 of the Sequence Listing or a19 bp spacer sequence from the sequences listed in SEQ ID NOs:1,410,430-1,410,472 of the Sequence Listing;

FIG. 1C is a plasmid (CTx-3) comprising yet another different codonoptimized gene for S. pyogenes Cas9 endonuclease. The CTx-3 plasmid alsocomprises a gRNA scaffold sequence, which includes a 20 bp spacersequence from the sequences listed in SEQ ID NOs: 1-467,030 of theSequence Listing or a 19 bp spacer sequence from the sequences listed inSEQ ID NOs: 1,410,430-1,410,472 of the Sequence Listing; and

FIG. 2A is a depiction of the type II CRISPR/Cas system.

FIG. 2B is a depiction of the type II CRISPR/Cas system.

FIG. 3A describes the cutting efficiency of S. pyogenes gRNAs in HEK293Ts targeting Exons 45, 51, and 53 of the dystrophin gene.

FIG. 3B describes the cutting efficiency of S. pyogenes gRNAs in HEK293Ts targeting Exons 55 and 70 of the dystrophin gene.

FIG. 4A describes the cutting efficiency of S. pyogenes gRNAs in HEK293Ts targeting the splice acceptor of Exons 43, 44, 45, 46, 50, 51, 52, 53and 55 of the dystrophin gene.

FIG. 4B describes the cutting efficiency of N. meningitides, S.thermophiles, and S. aureus gRNAs in HEK293 Ts targeting the spliceacceptor of Exons 43, 44, 45, 46, 50, 51, 52, 53 and 55 of thedystrophin gene.

FIG. 4C describes the cutting efficiency of Cpf1 gRNAs in HEK293 Tstargeting the splice acceptors of Exons 43, 44, 45, 46, 50, 51, 52, 53and 55 of the dystrophin gene.

FIGS. 5A-B describe cutting efficiencies and splice acceptor knock-outefficiencies of S. pyogenes gRNAs in HEK293 Ts targeting Exons 51, 45,53, 44, 46, 52, 50, 43, and 55 of the dystrophin gene.

FIG. 6 describes cutting efficiencies and splice acceptor knock-outefficiencies of N. meningitides (NM), S. thermophiles (ST), and S.aureus (SA) gRNAs in HEK293 Ts targeting Exons 51, 45, 53, 44, 46, 52,50, 43, and 55 of the dystrophin gene.

FIG. 7A describes the cutting efficiency of S. pyogenes gRNAs in HEK293Tcells where the gRNAs target the regions surrounding Exon 52 of thedystrophin gene.

FIG. 7B describes the cutting efficiency of S. pyogenes gRNAs in HEK293Tcells where the gRNAs target the regions surrounding Exons 44, 45, and54 of the dystrophin gene.

FIG. 8A describes the cutting efficiency of S. pyogenes gRNAs in iPSCswhere the gRNAs target the regions surrounding Exon 52 of the dystrophingene.

FIG. 8B describes the cutting efficiency of S. pyogenes gRNAs in iPSCswhere the gRNAs target the regions surrounding Exons 44, 45, and 54 ofthe dystrophin gene.

FIG. 9 describes the cutting efficiency comparison of S. pyogenes gRNAsin HEK293T cells and iPSCs where the gRNAs target the regionssurrounding Exons 44, 45, 52, and 54 of the dystrophin gene.

FIG. 10A, FIG. 10B, and FIG. 10C descibe clonal analysis of clonaldeletion events.

FIG. 11A and FIG. 11B describe sanger sequencing of Δ52 clones. SEQ IDNO: 1420000 corresponds to FIG. 11A. SEQ ID NO: 1420001 corresponds toFIG. 11B.

FIGS. 12A-E describe the cutting efficiencies of gRNAs selected via anin-vitro transcribed (IVT) gRNA screen.

FIG. 13A describes the homology directed repair (HDR) between Exon 45-55of the dystrophin gene.

FIG. 13B depicts the PCR confirmation of HDR at the Exon 45-55 locus ofthe dystrophin gene.

FIG. 14A depicts a three primer PCR assay.

FIG. 14B depicts results from the three primer PCR assay.

FIG. 14C describes data generated from the three primer PCR assay.

FIG. 15 describes 5 clones that have the desired Δ45-55 deletion. SEQ IDNO: 1420002 corresponds to clone 20. SEQ ID NO: 1420003 corresponds toclone 56. SEQ ID NO: 1420004 corresponds to clone 69. SEQ ID NO: 1420005corresponds to clone 74. SEQ ID NO: 1420006 corresponds to clone 100.

FIGS. 16A-B describes the SSEA-4 Staining and TRA-160 Staining resultsof the 5 clones that have the desired Δ45-55 deletion.

FIG. 17 depicts the expression of an internally deleted dystrophinprotein for all 5 edited clones.

FIG. 18 depicts myosin heavy chain staining of differentiated clone 56.

FIGS. 19A-19VV describe the results of a large scale lentiviral screen.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-467,030 is a list of gRNA 20 bp spacer sequences fortargeting the dystrophin gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 467,031-528,196 is a list of gRNA 20 bp spacer sequences fortargeting the dystrophin gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 528,197-553,198 is a list of gRNA 24 bp spacer sequences fortargeting the dystrophin gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 553,199-563,911 is a list of gRNA 24 bp spacer sequences fortargeting the dystrophin gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 563,912-627,854 is a list of gRNA 24 bp spacer sequences fortargeting the dystrophin gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 627,855-1,410,399 is a list of gRNA 20-24 bp spacersequences for targeting the dystrophin gene with an Acidominoccoccus, aLachnospiraceae, and a Franciscella Novicida Cpf1 endonuclease.

SEQ ID NOs: 1,410,400-1,410,402 is a list of gRNA 24 bp spacer sequencesfor targeting the dystrophin gene with a N. meningitides Cas9endonuclease.

SEQ ID NOs: 1,410,403-1,410,429 is a list of gRNA 23 bp spacer sequencesfor targeting the dystrophin gene with an Acidominoccoccus, aLachnospiraceae, and a Franciscella Novicida Cpf1 endonuclease.

SEQ ID NOs: 1,410,430-1,410,472 is a list of gRNA 19 bp spacer sequencesfor targeting the dystrophin gene with a S. pyogenes Cas9 endonuclease.

DETAILED DESCRIPTION

Duchenne Muscular Dystrophy (DMD)

DMD is caused by mutations in the dystrophin gene (Chromosome X:31,117,228-33,344,609 (Genome Reference Consortium—GRCh38/hg38)). With agenomic region of over 2.2 megabases in length, dystrophin is the secondlargest human gene. The dystrophin gene contains 79 exons that areprocessed into an 11,000 base pair mRNA that is translated into a 427kDa protein. Functionally, dystrophin acts as a linker between the actinfilaments and the extracellular matrix within muscle fibers. The Nterminus of dystrophin is an actin-binding domain, while the C terminusinteracts with a transmembrane scaffold that anchors the muscle fiber tothe extracellular matrix. Upon muscle contraction, dystrophin providesstructural support that allows the muscle tissue to withstand mechanicalforce. DMD is caused by a wide variety of mutations within thedystrophin gene that result in premature stop codons and therefore atruncated dystrophin protein. Truncated dystrophin proteins do notcontain the C terminus, and therefore cannot provide the structuralsupport necessary to withstand the stress of muscle contraction. As aresult, the muscle fibers pull themselves apart, which leads to musclewasting.

Therapeutic Approach

Provided herein are cellular, ex vivo and in vivo methods for usinggenome engineering tools to create permanent changes to the genome thatcan restore the dystrophin reading frame and restore dystrophin proteinactivity. Such methods use endonucleases, such as CRISPR/Cas9 nucleases,to permanently delete (excise), insert, or replace (delete and insert)exons (i.e., mutations in the coding and/or splicing sequences) in thegenomic locus of the dystrophin gene. In this way, the present inventionmimics the product produced by exon skipping, and/or restores thereading frame with as few as a single treatment (rather than deliverexon skipping oligos for the lifetime of the patient). Pre-clinicalstudies have been performed regarding expression of the C terminus ofdystrophin by making targeted changes to the genome using Zinc-Finger-,TALE-, and CRISPR/Cas9-based nucleases. In one example, a large genomicregion was deleted that is projected to treat over 60% of the patientswith DMD.

Provided herein are methods for treating a patient with DMD. An exampleof such method is an ex vivo cell based therapy. For example, a DMDpatient specific iPS cell line is created. Then, the chromosomal DNA ofthese iPS cells is corrected using the materials and methods describedherein. Next, the corrected iPSCs are differentiated into Pax7+ muscleprogenitor cells. Finally, the progenitor cells are implanted into thepatient. There are many advantages to this ex vivo approach.

One advantage of an ex vivo cell therapy approach is the ability toconduct a comprehensive analysis of the therapeutic prior toadministration. All nuclease based therapeutics have some level ofoff-target effects. Performing gene correction ex vivo allows one tofully characterize the corrected cell population prior to implantation.Aspects of the present disclosure include sequencing the entire genomeof the corrected cells to ensure that the off-target cuts, if any, arein genomic locations associated with minimal risk to the patient.Furthermore, clonal populations of cells can be isolated prior toimplantation.

Another advantage of ex vivo cell therapy relates to genetic correctionin iPSCs compared to other primary cell sources. iPSCs are prolific,making it easy to obtain the large number of cells that will be requiredfor a cell based therapy. Furthermore, iPSCs are an ideal cell type forperforming clonal isolations. This allows screening for the correctgenomic correction, without risking a decrease in viability. Incontrast, other potential cell types, such as primary myoblasts, areviable for only a few passages and difficult to clonally expand. Also,patient specific DMD myoblasts will be unhealthy due to the lack ofdystrophin protein. On the other hand, patient derived DMD iPSCs willnot display a diseased phenotype, as they do not express dystrophin inthis differentiation state. Therefore, manipulation of DMD iPSCs will bemuch easier, and will shorten the amount of time needed to make thedesired genetic correction.

A further advantage of ex vivo cell therapy relates to the implantationof myogenic Pax7+ progenitors versus myoblasts. Pax7+ cells are acceptedas myogenic satellite cells. Pax7+ progenitors are mono-nuclear cellsthat sit on the periphery of the multi-nucleated muscle fibers. Inresponse to injury, the progenitors divide and fuse to the existingfibers. In contrast, myoblasts fuse directly to the muscle fiber uponimplantation and have minimal proliferative capacity in vivo. Therefore,myoblasts cannot aid in healing following repeated injury, while Pax7+progenitors can function as a reservoir and help heal the muscle for thelifetime of the patient.

Another example of such method is an in vivo based therapy. In thismethod, the chromosomal DNA of the cells in the patient is correctedusing the materials and methods described herein.

The advantage of in vivo gene therapy is the ease of therapeuticproduction and administration. The same therapeutic cocktail will havethe potential to reach a subset of the DMD patient population (n>1). Incontrast, the ex vivo cell therapy proposed requires a customtherapeutic to be developed for each patient (n=1). Ex vivo cell therapydevelopment requires time, which certain advanced DMD patients may nothave.

Also provided herein is a cellular method for editing the dystrophingene in a human cell by genome editing. For example, a cell is isolatedfrom a patient or animal. Then, the chromosomal DNA of the cell iscorrected using the materials and methods described herein.

A number of types of genomic target sites can be present in addition tomutations in the coding and splicing sequences.

The regulation of transcription and translation implicates a number ofdifferent classes of sites that interact with cellular proteins ornucleotides. Often the DNA binding sites of transcription factors orother proteins can be targeted for mutation or deletion to study therole of the site, though they can also be targeted to change geneexpression. Sites can be added through non-homologous end joining (NHEJ)or direct genome editing by homology directed repair (HDR). Increaseduse of genome sequencing, RNA expression and genome-wide studies oftranscription factor binding have increased our ability to identify howthe sites lead to developmental or temporal gene regulation. Thesecontrol systems can be direct or can involve extensive cooperativeregulation that can require the integration of activities from multipleenhancers. Transcription factors typically bind 6-12 bp-long degenerateDNA sequences. The low level of specificity provided by individual sitessuggests that complex interactions and rules are involved in binding andthe functional outcome. Binding sites with less degeneracy can providesimpler means of regulation. Artificial transcription factors can bedesigned to specify longer sequences that have less similar sequences inthe genome and have lower potential for off-target cleavage. Any ofthese types of binding sites can be mutated, deleted or even created toenable changes in gene regulation or expression (Canver, M. C. et al.,Nature (2015)).

Another class of gene regulatory regions having these features ismicroRNA (miRNA) binding sites. miRNAs are non-coding RNAs that play keyroles in post-transcriptional gene regulation. miRNA can regulate theexpression of 30% of all mammalian protein-encoding genes. Specific andpotent gene silencing by double stranded RNA (RNAi) was discovered, plusadditional small noncoding RNA (Canver, M. C. et al., Nature (2015)).The largest class of noncoding RNAs important for gene silencing aremiRNAs. In mammals, miRNAs are first transcribed as long RNAtranscripts, which can be separate transcriptional units, part ofprotein introns, or other transcripts. The long transcripts are calledprimary miRNA (pri-miRNA) that include imperfectly base-paired hairpinstructures. These pri-miRNAs can be cleaved into one or more shorterprecursor miRNAs (pre-miRNAs) by Microprocessor, a protein complex inthe nucleus, involving Drosha.

Pre-miRNAs are short stem loops ˜70 nucleotides in length with a2-nucleotide 3′-overhang that are exported, into the mature 19-25nucleotide miRNA:miRNA* duplexes. The miRNA strand with lower basepairing stability (the guide strand) can be loaded onto the RNA-inducedsilencing complex (RISC). The passenger guide strand (marked with *),can be functional, but is usually degraded. The mature miRNA tethersRISC to partly complementary sequence motifs in target mRNAspredominantly found within the 3′ untranslated regions (UTRs) andinduces posttranscriptional gene silencing (Bartel, D. P. Cell 136,215-233 (2009); Saj, A. & Lai, E. C. Curr Opin Genet Dev 21, 504-510(2011)).

miRNAs can be important in development, differentiation, cell cycle andgrowth control, and in virtually all biological pathways in mammals andother multicellular organisms. miRNAs can also be involved in cell cyclecontrol, apoptosis and stem cell differentiation, hematopoiesis,hypoxia, muscle development, neurogenesis, insulin secretion,cholesterol metabolism, aging, viral replication and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, whilean individual transcript can be targeted by many different miRNAs. Morethan 28645 microRNAs have been annotated in the latest release ofmiRBase (v.21). Some miRNAs can be encoded by multiple loci, some ofwhich can be expressed from tandemly co-transcribed clusters. Thefeatures allow for complex regulatory networks with multiple pathwaysand feedback controls. miRNAs can be integral parts of these feedbackand regulatory circuits and can help regulate gene expression by keepingprotein production within limits (Herranz, H. & Cohen, S. M. Genes Dev24, 1339-1344 (2010); Posadas, D. M. & Carthew, R. W. Curr Opin GenetDev 27, 1-6 (2014)).

miRNA can also be important in a large number of human diseases that areassociated with abnormal miRNA expression. This association underscoresthe importance of the miRNA regulatory pathway. Recent miRNA deletionstudies have linked miRNA with regulation of the immune responses(Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

miRNA also have a strong link to cancer and can play a role in differenttypes of cancer. miRNAs have been found to be downregulated in a numberof tumors. miRNA can be important in the regulation of keycancer-related pathways, such as cell cycle control and the DNA damageresponse, and can therefore be used in diagnosis and can be targetedclinically. MicroRNAs can delicately regulate the balance ofangiogenesis, such that experiments depleting all microRNAs suppresstumor angiogenesis (Chen, S. et al., Genes Dev 28, 1054-1067 (2014)).

As has been shown for protein coding genes, miRNA genes can also besubject to epigenetic changes occurring with cancer. Many miRNA loci canbe associated with CpG islands increasing their opportunity forregulation by DNA methylation (Weber, B., Stresemann, C., Brueckner, B.& Lyko, F. Cell Cycle 6, 1001-1005 (2007)). The majority of studies haveused treatment with chromatin remodeling drugs to reveal epigeneticallysilenced miRNAs.

In addition to their role in RNA silencing, miRNA can also activatetranslation (Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev 27, 1-6(2014)). Knocking out these sites can lead to decreased expression ofthe targeted gene, while introducing these sites can increaseexpression.

Individual miRNA can be knocked out most effectively by mutating theseed sequence (bases 2-8 of the microRNA), which can be important forbinding specificity. Cleavage in this region, followed by mis-repair byNHEJ can effectively abolish miRNA function by blocking binding totarget sites. miRNA could also be inhibited by specific targeting of thespecial loop region adjacent to the palindromic sequence. Catalyticallyinactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. etal., Sci Rep 4, 3943 (2014)). In addition to targeting the miRNA, thebinding sites can also be targeted and mutated to prevent the silencingby miRNA.

Human Cells

For ameliorating DMD, as described and illustrated herein, the principaltargets for gene editing are human cells. For example, in the ex vivomethods, the human cells can be somatic cells, which after beingmodified using the techniques as described, can give rise to Pax7+muscle progenitor cells. For example, in the in vivo methods, the humancells can be muscle cells or muscle precursor cells.

By performing gene editing in autologous cells that are derived from andtherefore already completely matched with the patient in need, it ispossible to generate cells that can be safely re-introduced into thepatient, and effectively give rise to a population of cells that can beeffective in ameliorating one or more clinical conditions associatedwith the patient's disease.

Progenitor cells (also referred to as stem cells herein) are capable ofboth proliferation and giving rise to more progenitor cells, these inturn having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one aspect, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell can derive from amultipotent cell that itself is derived from a multipotent cell, and soon. While each of these multipotent cells can be considered stem cells,the range of cell types that each can give rise to can varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity can benatural or may be induced artificially upon treatment with variousfactors. In many biological instances, stem cells can be also“multipotent” because they can produce progeny of more than one distinctcell type, but this is not required for “stem-ness.”

Self-renewal can be another important aspect of the stem cell. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells can divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Generally,“progenitor cells” have a cellular phenotype that is more primitive(i.e., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell). Often, progenitorcells also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellto which it is being compared. Thus, stem cells can differentiate intolineage-restricted precursor cells (such as a myocyte progenitor cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a myocyte precursor), and then to anend-stage differentiated cell, such as a myocyte, which plays acharacteristic role in a certain tissue type, and may or may not retainthe capacity to proliferate further.

Induced Pluripotent Stem Cells

In some examples, the genetically engineered human cells describedherein can be induced pluripotent stem cells (iPSCs). An advantage ofusing iPSCs is that the cells can be derived from the same subject towhich the progenitor cells are to be administered. That is, a somaticcell can be obtained from a subject, reprogrammed to an inducedpluripotent stem cell, and then re-differentiated into a progenitor cellto be administered to the subject (e.g., autologous cells). Because theprogenitors are essentially derived from an autologous source, the riskof engraftment rejection or allergic response can be reduced compared tothe use of cells from another subject or group of subjects. In addition,the use of iPSCs negates the need for cells obtained from an embryonicsource. Thus, in one aspect, the stem cells used in the disclosedmethods are not embryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to iPSCs. Exemplary methods are known to those of skill inthe art and are described briefly herein below.

The term “reprogramming” refers to a process that alters or reverses thedifferentiation state of a differentiated cell (e.g., a somatic cell).Stated another way, reprogramming refers to a process of driving thedifferentiation of a cell backwards to a more undifferentiated or moreprimitive type of cell. It should be noted that placing many primarycells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such cells included in the termdifferentiated cells does not render these cells non-differentiatedcells (e.g., undifferentiated cells) or pluripotent cells. Thetransition of a differentiated cell to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. Reprogramming encompassescomplete reversion of the differentiation state of a differentiated cell(e.g., a somatic cell) to a pluripotent state or a multipotent state.Reprogramming can encompass complete or partial reversion of thedifferentiation state of a differentiated cell (e.g., a somatic cell) toan undifferentiated cell (e.g., an embryonic-like cell). Reprogrammingcan result in expression of particular genes by the cells, theexpression of which further contributes to reprogramming. In certainexamples described herein, reprogramming of a differentiated cell (e.g.,a somatic cell) can cause the differentiated cell to assume anundifferentiated state (e.g., is an undifferentiated cell). Theresulting cells are referred to as “reprogrammed cells,” or “inducedpluripotent stem cells (iPSCs or iPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a myogenic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some examples.

Many methods are known in the art that can be used to generatepluripotent stem cells from somatic cells. Any such method thatreprograms a somatic cell to the pluripotent phenotype would beappropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described. Mousesomatic cells can be converted to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76(2006). iPSCs resemble ES cells, as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germ line transmission [see, e.g., Maherali and Hochedlinger,Cell Stem Cell. 3(6):595-605 (2008)], and tetraploid complementation.

Human iPSCs can be obtained using similar transduction methods, and thetranscription factor trio, OCT4, SOX2, and NANOG, has been establishedas the core set of transcription factors that govern pluripotency; see,e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57(2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121(2014); Focosi et al., Blood Cancer Journal 4: e211 (2014); andreferences cited therein. The production of iPSCs can be achieved by theintroduction of nucleic acid sequences encoding stem cell-associatedgenes into an adult, somatic cell, historically using viral vectors.

iPSCs can be generated or derived from terminally differentiated somaticcells, as well as from adult stem cells, or somatic stem cells. That is,a non-pluripotent progenitor cell can be rendered pluripotent ormultipotent by reprogramming. In such instances, it may not be necessaryto include as many reprogramming factors as required to reprogram aterminally differentiated cell. Further, reprogramming can be induced bythe non-viral introduction of reprogramming factors, e.g., byintroducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Reprogramming can beachieved by introducing a combination of nucleic acids encoding stemcell-associated genes, including, for example, Oct-4 (also known asOct-3/4 or Pouf51), SoxI, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klfl, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28.Reprogramming using the methods and compositions described herein canfurther comprise introducing one or more of Oct-3/4, a member of the Soxfamily, a member of the Klf family, and a member of the Myc family to asomatic cell. The methods and compositions described herein can furthercomprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYCand Klf4 for reprogramming. As noted above, the exact method used forreprogramming is not necessarily critical to the methods andcompositions described herein. However, where cells differentiated fromthe reprogrammed cells are to be used in, e.g., human therapy, in oneaspect the reprogramming is not effected by a method that alters thegenome. Thus, in such examples, reprogramming can be achieved, e.g.,without the use of viral or plasmid vectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various agents, e.g., small molecules, as shown by Shi etal., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., NatureBiotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Thus, an agent or combination of agents that enhance theefficiency or rate of induced pluripotent stem cell production can beused in the production of patient-specific or disease-specific iPSCs.Some non-limiting examples of agents that enhance reprogrammingefficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9ahistone methyltransferase), PD0325901 (a MEK inhibitor), DNAmethyltransferase inhibitors, histone deacetylase (HDAC) inhibitors,valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide,hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-IH,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzam ides (e.g.,CI-994 (e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824,CBHA (m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199,Tubacin, A-161906, proxamide, oxamflatin, 3-CI-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogrammingenhancing agents include, for example, dominant negative forms of theHDACs (e.g., catalytically inactive forms), siRNA inhibitors of theHDACs, and antibodies that specifically bind to the HDACs. Suchinhibitors are available, e.g., from BIOMOL International, Fukasawa,Merck Biosciences, Novartis, Gloucester Pharmaceuticals, TitanPharmaceuticals, MethylGene, and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto,Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, acell that expresses Oct4 or Nanog is identified as pluripotent. Methodsfor detecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses.Detection can involve, not only RT-PCR, but can also include detectionof protein markers. Intracellular markers can be best identified viaRT-PCR, or protein detection methods such as immunocytochemistry, whilecell surface markers are readily identified, e.g., byimmunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate into cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells can be introduced into nude mice andhistology and/or immunohistochemistry can be performed on a tumorarising from the cells. The growth of a tumor comprising cells from allthree germ layers, for example, further indicates that the cells arepluripotent stem cells.

Creating DMD Patient Specific iPSCs

One step of the ex vivo methods of the present disclosure can involvecreating a DMD patient specific iPS cell, DMD patient specific iPScells, or a DMD patient specific iPS cell line. There are manyestablished methods in the art for creating patient specific iPS cells,as described in Takahashi and Yamanaka 2006; Takahashi, Tanabe et al.2007. In addition, differentiation of pluripotent cells toward themuscle lineage can be accomplished by technology developed by AnagenesisBiotechnologies, as described in International patent applicationpublication numbers WO2013/030243 and WO2012/101114. For example, thecreating step can comprise: a) isolating a somatic cell, such as a skincell or fibroblast from the patient; and b) introducing a set ofpluripotency-associated genes into the somatic cell in order to inducethe cell to become a pluripotent stem cell. The set ofpluripotency-associated genes can be one or more of the genes selectedfrom the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC.

Genome Editing

Genome editing generally refers to the process of modifying thenucleotide sequence of a genome, preferably in a precise orpre-determined manner. Examples of methods of genome editing describedherein include methods of using site-directed nucleases to cutdeoxyribonucleic acid (DNA) at precise target locations in the genome,thereby creating single-strand or double-strand DNA breaks at particularlocations within the genome. Such breaks can be and regularly arerepaired by natural, endogenous cellular processes, such ashomology-directed repair (HDR) and non-homologous end-joining (NHEJ), asrecently reviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015).NHEJ directly joins the DNA ends resulting from a double-strand break,sometimes with the loss or addition of nucleotide sequence, which candisrupt or enhance gene expression. HDR utilizes a homologous sequence,or donor sequence, as a template for inserting a defined DNA sequence atthe break point. The homologous sequence can be in the endogenousgenome, such as a sister chromatid. Alternatively, the donor can be anexogenous nucleic acid, such as a plasmid, a single-strandoligonucleotide, a double-strand oligonucleotide, a duplexoligonucleotide or a virus, that has regions of high homology with thenuclease-cleaved locus, but which can also contain additional sequenceor sequence changes including deletions that can be incorporated intothe cleaved target locus. A third repair mechanism can bemicrohomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ”, in which the genetic outcome is similar to NHEJ inthat small deletions and insertions can occur at the cleavage site. MMEJcan make use of homologous sequences of a few basepairs flanking the DNAbreak site to drive a more favored DNA end joining repair outcome, andrecent reports have further elucidated the molecular mechanism of thisprocess; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015); Kentet al., Nature Structural and Molecular Biology, Adv. Onlinedoi:10.1038/nsmb.2961(2015); Mateos-Gomez et al., Nature 518, 254-57(2015); Ceccaldi et al., Nature 528, 258-62 (2015). In some instances itmay be possible to predict likely repair outcomes based on analysis ofpotential microhomologies at the site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenomic alterations. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as close as near to thesite of intended mutation. This can be achieved via the use ofsite-directed polypeptides, as described and illustrated herein.

Site-directed polypeptides, such as a DNA endonuclease, can introducedouble-strand breaks or single-strand breaks in nucleic acids, e.g.,genomic DNA. The double-strand break can stimulate a cell's endogenousDNA-repair pathways (e.g., homology-dependent repair or non-homologousend joining or alternative non-homologous end joining (A-NHEJ) ormicrohomology-mediated end joining). NHEJ can repair cleaved targetnucleic acid without the need for a homologous template. This cansometimes result in small deletions or insertions (indels) in the targetnucleic acid at the site of cleavage, and can lead to disruption oralteration of gene expression. HDR can occur when a homologous repairtemplate, or donor, is available. The homologous donor template cancomprise sequences that can be homologous to sequences flanking thetarget nucleic acid cleavage site. The sister chromatid can be used bythe cell as the repair template. However, for the purposes of genomeediting, the repair template can be supplied as an exogenous nucleicacid, such as a plasmid, duplex oligonucleotide, single-strandoligonucleotide, double-stranded oligonucleotide, or viral nucleic acid.With exogenous donor templates, an additional nucleic acid sequence(such as a transgene) or modification (such as a single or multiple basechange or a deletion) can be introduced between the flanking regions ofhomology so that the additional or altered nucleic acid sequence alsobecomes incorporated into the target locus. MMEJ can result in a geneticoutcome that is similar to NHEJ in that small deletions and insertionscan occur at the cleavage site. MMEJ can make use of homologoussequences of a few basepairs flanking the cleavage site to drive afavored end-joining DNA repair outcome. In some instances it may bepossible to predict likely repair outcomes based on analysis ofpotential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence or polynucleotide donortemplate) herein. The donor polynucleotide, a portion of the donorpolynucleotide, a copy of the donor polynucleotide, or a portion of acopy of the donor polynucleotide can be inserted into the target nucleicacid cleavage site. The donor polynucleotide can be an exogenouspolynucleotide sequence, i.e., a sequence that does not naturally occurat the target nucleic acid cleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)genomic locus can be found in the genomes of many prokaryotes (e.g.,bacteria and archaea). In prokaryotes, the CRISPR locus encodes productsthat function as a type of immune system to help defend the prokaryotesagainst foreign invaders, such as virus and phage. There are threestages of CRISPR locus function: integration of new sequences into theCRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreigninvader nucleic acid. Five types of CRISPR systems (e.g., Type I, TypeII, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referredto as “repeats.” When expressed, the repeats can form secondarystructures (e.g., hairpins) and/or comprise unstructured single-strandedsequences. The repeats usually occur in clusters and frequently divergebetween species. The repeats are regularly interspaced with uniqueintervening sequences referred to as “spacers,” resulting in arepeat-spacer-repeat locus architecture. The spacers are identical to orhave high homology with known foreign invader sequences. A spacer-repeatunit encodes a crisprRNA (crRNA), which is processed into a mature formof the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequencethat is involved in targeting a target nucleic acid (in the naturallyoccurring form in prokaryotes, the spacer sequence targets the foreigninvader nucleic acid). A spacer sequence is located at the 5′ or 3′ endof the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPRAssociated (Cas) genes. Cas genes encode endonucleases involved in thebiogenesis and the interference stages of crRNA function in prokaryotes.Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires atrans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified byendogenous RNaseIII, and then hybridizes to a crRNA repeat in thepre-crRNA array. Endogenous RNaseIII can be recruited to cleave thepre-crRNA. Cleaved crRNAs can be subjected to exoribonuclease trimmingto produce the mature crRNA form (e.g., 5′ trimming). The tracrRNA canremain hybridized to the crRNA, and the tracrRNA and the crRNA associatewith a site-directed polypeptide (e.g., Cas9). The crRNA of thecrRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleicacid to which the crRNA can hybridize. Hybridization of the crRNA to thetarget nucleic acid can activate Cas9 for targeted nucleic acidcleavage. The target nucleic acid in a Type II CRISPR system is referredto as a protospacer adjacent motif (PAM). In nature, the PAM isessential to facilitate binding of a site-directed polypeptide (e.g.,Cas9) to the target nucleic acid. Type II systems (also referred to asNmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B(CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed thatthe CRISPR/Cas9 system is useful for RNA-programmable genome editing,and international patent application publication number WO2013/176772provides numerous examples and applications of the CRISPR/Casendonuclease system for site-specific gene editing.

Type V CRISPR Systems

Type V CRISPR systems have several important differences from Type IIsystems. For example, Cpf1 is a single RNA-guided endonuclease that, incontrast to Type II systems, lacks tracrRNA. In fact, Cpf1-associatedCRISPR arrays can be processed into mature crRNAs without therequirement of an additional trans-activating tracrRNA. The Type VCRISPR array can be processed into short mature crRNAs of 42-44nucleotides in length, with each mature crRNA beginning with 19nucleotides of direct repeat followed by 23-25 nucleotides of spacersequence. In contrast, mature crRNAs in Type II systems can start with20-24 nucleotides of spacer sequence followed by about 22 nucleotides ofdirect repeat. Also, Cpf1 can utilize a T-rich protospacer-adjacentmotif such that Cpf1-crRNA complexes efficiently cleave target DNApreceded by a short T-rich PAM, which is in contrast to the G-rich PAMfollowing the target DNA for Type II systems. Thus, Type V systemscleave at a point that is distant from the PAM, while Type II systemscleave at a point that is adjacent to the PAM. In addition, in contrastto Type II systems, Cpf1 cleaves DNA via a staggered DNA double-strandedbreak with a 4 or 5 nucleotide 5′ overhang. Type II systems cleave via ablunt double-stranded break. Similar to Type II systems, Cpf1 contains apredicted RuvC-like endonuclease domain, but lacks a second HNHendonuclease domain, which is in contrast to Type II systems.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides in FIG.1 of Fonfara et al., Nucleic Acids Research, 42: 2577-2590 (2014). TheCRISPR/Cas gene naming system has undergone extensive rewriting sincethe Cas genes were discovered. FIG. 5 of Fonfara, supra, provides PAMsequences for the Cas9 polypeptides from various species.

Site-Directed Polypeptides

A site-directed polypeptide is a nuclease used in genome editing tocleave DNA. The site-directed can be administered to a cell or a patientas either: one or more polypeptides, or one or more mRNAs encoding thepolypeptide.

In the context of a CRISPR/Cas or CRISPR/Cpf1 system, the site-directedpolypeptide can bind to a guide RNA that, in turn, specifies the site inthe target DNA to which the polypeptide is directed. In the CRISPR/Casor CRISPR/Cpf1 systems disclosed herein, the site-directed polypeptidecan be an endonuclease, such as a DNA endonuclease.

A site-directed polypeptide can comprise a plurality of nucleicacid-cleaving (i.e., nuclease) domains. Two or more nucleicacid-cleaving domains can be linked together via a linker. For example,the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type Cas9 enzymes comprise two nucleasedomains, a HNH nuclease domain and a RuvC domain. Herein, the “Cas9”refers to both naturally-occurring and recombinant Cas9s. Cas9 enzymescontemplated herein can comprise a HNH or HNH-like nuclease domain,and/or a RuvC or RuvC-like nuclease domain.

HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-likedomains comprises two antiparallel β-strands and an α-helix. HNH orHNH-like domains comprises a metal binding site (e.g., a divalent cationbinding site). HNH or HNH-like domains can cleave one strand of a targetnucleic acid (e.g., the complementary strand of the crRNA targetedstrand).

RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold.RuvC/RNaseH domains are involved in a diverse set of nucleic acid-basedfunctions including acting on both RNA and DNA. The RNaseH domaincomprises 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site(e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-likedomains can cleave one strand of a target nucleic acid (e.g., thenon-complementary strand of a double-stranded target DNA).

Site-directed polypeptides can introduce double-strand breaks orsingle-strand breaks in nucleic acids, e.g., genomic DNA. Thedouble-strand break can stimulate a cell's endogenous DNA-repairpathways (e.g., homology-dependent repair (HDR) or non-homologous endjoining (NHEJ) or alternative non-homologous end joining (A-NHEJ) ormicrohomology-mediated end joining (MMEJ)). NHEJ can repair cleavedtarget nucleic acid without the need for a homologous template. This cansometimes result in small deletions or insertions (indels) in the targetnucleic acid at the site of cleavage, and can lead to disruption oralteration of gene expression. HDR can occur when a homologous repairtemplate, or donor, is available. The homologous donor template cancomprise sequences that are homologous to sequences flanking the targetnucleic acid cleavage site. The sister chromatid can be used by the cellas the repair template. However, for the purposes of genome editing, therepair template can be supplied as an exogenous nucleic acid, such as aplasmid, duplex oligonucleotide, single-strand oligonucleotide or viralnucleic acid. With exogenous donor templates, an additional nucleic acidsequence (such as a transgene) or modification (such as a single ormultiple base change or a deletion) can be introduced between theflanking regions of homology so that the additional or altered nucleicacid sequence also becomes incorporated into the target locus. MMEJ canresult in a genetic outcome that is similar to NHEJ in that smalldeletions and insertions can occur at the cleavage site. MMEJ can makeuse of homologous sequences of a few basepairs flanking the cleavagesite to drive a favored end-joining DNA repair outcome. In someinstances it may be possible to predict likely repair outcomes based onanalysis of potential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination is used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence) herein. The donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the target nucleic acid cleavage site. The donorpolynucleotide can be an exogenous polynucleotide sequence, i.e., asequence that does not naturally occur at the target nucleic acidcleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

The site-directed polypeptide can comprise an amino acid sequence havingat least 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% amino acidsequence identity to a wild-type exemplary site-directed polypeptide[e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence ID No. 8 orSapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], andvarious other site-directed polypeptides).

The site-directed polypeptide comprises at least 70, 75, 80, 85, 90, 95,97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g.,Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. Thesite-directed polypeptide can comprise at most: 70, 75, 80, 85, 90, 95,97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g.,Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. Thesite-directed polypeptide can comprise at least: 70, 75, 80, 85, 90, 95,97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g.,Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNHnuclease domain of the site-directed polypeptide. The site-directedpolypeptide can comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or100% identity to a wild-type site-directed polypeptide (e.g., Cas9 fromS. pyogenes, supra) over 10 contiguous amino acids in a HNH nucleasedomain of the site-directed polypeptide. The site-directed polypeptidecan comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identityto a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids in a RuvC nuclease domain of thesite-directed polypeptide. The site-directed polypeptide comprises atmost: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide.

The site-directed polypeptide can comprise a modified form of awild-type exemplary site-directed polypeptide. The modified form of thewild-type exemplary site-directed polypeptide can comprise a mutationthat reduces the nucleic acid-cleaving activity of the site-directedpolypeptide. The modified form of the wild-type exemplary site-directedpolypeptide can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form ofthe site-directed polypeptide can have no substantial nucleicacid-cleaving activity. When a site-directed polypeptide is a modifiedform that has no substantial nucleic acid-cleaving activity, it isreferred to herein as “enzymatically inactive.”

The modified form of the site-directed polypeptide can comprise amutation such that it can induce a single-strand break (SSB) on a targetnucleic acid (e.g., by cutting only one of the sugar-phosphate backbonesof a double-strand target nucleic acid). The mutation can result in lessthan 90%, less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, less than 10%, less than5%, or less than 1% of the nucleic acid-cleaving activity in one or moreof the plurality of nucleic acid-cleaving domains of the wild-type sitedirected polypeptide (e.g., Cas9 from S. pyogenes, supra). The mutationcan result in one or more of the plurality of nucleic acid-cleavingdomains retaining the ability to cleave the complementary strand of thetarget nucleic acid, but reducing its ability to cleave thenon-complementary strand of the target nucleic acid. The mutation canresult in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the non-complementary strand of thetarget nucleic acid, but reducing its ability to cleave thecomplementary strand of the target nucleic acid. For example, residuesin the wild-type exemplary S. pyogenes Cas9 polypeptide, such as Asp10,His840, Asn854 and Asn856, are mutated to inactivate one or more of theplurality of nucleic acid-cleaving domains (e.g., nuclease domains). Theresidues to be mutated can correspond to residues Asp10, His840, Asn854and Asn856 in the wild-type exemplary S. pyogenes Cas9 polypeptide(e.g., as determined by sequence and/or structural alignment).Non-limiting examples of mutations include D10A, H840A, N854A or N856A.One skilled in the art will recognize that mutations other than alaninesubstitutions can be suitable.

A D10A mutation can be combined with one or more of H840A, N854A, orN856A mutations to produce a site-directed polypeptide substantiallylacking DNA cleavage activity. A H840A mutation can be combined with oneor more of D10A, N854A, or N856A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. A N854Amutation can be combined with one or more of H840A, D10A, or N856Amutations to produce a site-directed polypeptide substantially lackingDNA cleavage activity. A N856A mutation can be combined with one or moreof H840A, N854A, or D10A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. Site-directedpolypeptides that comprise one substantially inactive nuclease domainare referred to as “nickases”.

Nickase variants of RNA guided endonucleases, for example Cas9, can beused to increase the specificity of CRISPR-mediated genome editing. Wildtype Cas9 is typically guided by a single guide RNA designed tohybridize with a specified ˜20 nucleotide sequence in the targetsequence (such as an endogenous genomic locus). However, severalmismatches can be tolerated between the guide RNA and the target locus,effectively reducing the length of required homology in the target siteto, for example, as little as 13 nt of homology, and thereby resultingin elevated potential for binding and double-strand nucleic acidcleavage by the CRISPR/Cas9 complex elsewhere in the target genome—alsoknown as off-target cleavage. Because nickase variants of Cas9 each onlycut one strand, in order to create a double-strand break it is necessaryfor a pair of nickases to bind in close proximity and on oppositestrands of the target nucleic acid, thereby creating a pair of nicks,which is the equivalent of a double-strand break. This requires that twoseparate guide RNAs—one for each nickase—must bind in close proximityand on opposite strands of the target nucleic acid. This requirementessentially doubles the minimum length of homology needed for thedouble-strand break to occur, thereby reducing the likelihood that adouble-strand cleavage event will occur elsewhere in the genome, wherethe two guide RNA sites—if they exist—are unlikely to be sufficientlyclose to each other to enable the double-strand break to form. Asdescribed in the art, nickases can also be used to promote HDR versusNHEJ. HDR can be used to introduce selected changes into target sites inthe genome through the use of specific donor sequences that effectivelymediate the desired changes. Descriptions of various CRISPR/Cas systemsfor use in gene editing can be found, e.g., in international patentapplication publication number WO2013/176772, and in NatureBiotechnology 32, 347-355 (2014), and references cited therein.

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagines, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation can convert the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

The site-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive site-directedpolypeptide) can target nucleic acid. The site-directed polypeptide(e.g., variant, mutated, enzymatically inactive and/or conditionallyenzymatically inactive endoribonuclease) can target DNA. The sitedirected polypeptide (e.g., variant, mutated, enzymatically inactiveand/or conditionally enzymatically inactive endoribonuclease) can targetRNA.

The site-directed polypeptide can comprise one or more non-nativesequences (e.g., the site-directed polypeptide is a fusion protein).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acidcleaving domains (i.e., a HNH domain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein oneor both of the nucleic acid cleaving domains comprise at least 50% aminoacid identity to a nuclease domain from Cas9 from a bacterium (e.g., S.pyogenes).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), and non-native sequence (for example, anuclear localization signal) or a linker linking the site-directedpolypeptide to a non-native sequence.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein the site-directed polypeptidecomprises a mutation in one or both of the nucleic acid cleaving domainsthat reduces the cleaving activity of the nuclease domains by at least50%.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein one of the nuclease domains comprisesa mutation of aspartic acid 10, and/or wherein one of the nucleasedomains can comprise a mutation of histidine 840, and wherein themutation reduces the cleaving activity of the nuclease domain(s) by atleast 50%.

The one or more site-directed polypeptides, e.g. DNA endonucleases, cancomprise two nickases that together effect one double-strand break at aspecific locus in the genome, or four nickases that together effect orcause two double-strand breaks at specific loci in the genome.Alternatively, one site-directed polypeptide, e.g. DNA endonuclease, caneffect or cause one double-strand break at a specific locus in thegenome.

Genome-Targeting Nucleic Acid

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. The genome-targeting nucleic acid can be an RNA. Agenome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. Aguide RNA can comprise at least a spacer sequence that hybridizes to atarget nucleic acid sequence of interest, and a CRISPR repeat sequence.In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeatsequence and tracrRNA sequence hybridize to each other to form a duplex.In the Type V guide RNA (gRNA), the crRNA forms a duplex. In bothsystems, the duplex can bind a site-directed polypeptide, such that theguide RNA and site-direct polypeptide form a complex. Thegenome-targeting nucleic acid can provide target specificity to thecomplex by virtue of its association with the site-directed polypeptide.The genome-targeting nucleic acid thus can direct the activity of thesite-directed polypeptide.

Exemplary guide RNAs include the spacer sequences in the SequenceListing, shown with the genome location of their target sequence, whichis within or near the dystrophin gene, and the associated Cas9 cut site,wherein the genome location is based on the GRCh38/hg38 human genomeassembly.

Each guide RNA can be designed to include a spacer sequencecomplementary to its genomic target sequence, which is within or nearthe dystrophin gene. For example, each of the spacer sequences in theSequence Listing can be put into a single strand guide RNA (sgRNA)(e.g., an RNA chimera) or a crRNA (along with a corresponding tracrRNA).See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al.,Nature, 471, 602-607 (2011).

The genome-targeting nucleic acid can be a double-molecule guide RNA.The genome-targeting nucleic acid can be a single-molecule guide RNA.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) in a Type II system can comprise, inthe 5′ to 3′ direction, an optional spacer extension sequence, a spacersequence, a minimum CRISPR repeat sequence, a single-molecule guidelinker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence. The optional tracrRNA extensioncan comprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

A single-molecule guide RNA (sgRNA) in a Type V system can comprise, inthe 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacersequence.

By way of illustration, guide RNAs used in the CRISPR/Cas/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating RNAs of greater length isto produce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 of Cpf1 endonuclease, are morereadily generated enzymatically. Various types of RNA modifications canbe introduced during or after chemical synthesis and/or enzymaticgeneration of RNAs, e.g., modifications that enhance stability, reducethe likelihood or degree of innate immune response, and/or enhance otherattributes, as described in the art.

Spacer Extension Sequence

In some examples of genome-targeting nucleic acids, a spacer extensionsequence can modify activity, provide stability and/or provide alocation for modifications of a genome-targeting nucleic acid. A spacerextension sequence can modify on or off target activity or specificity.In some examples, a spacer extension sequence can be provided. Thespacer extension sequence can have a length of more than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000,4000, 5000, 6000, or 7000 or more nucleotides. The spacer extensionsequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000,7000 or more nucleotides. The spacer extension sequence can be less than10 nucleotides in length. The spacer extension sequence can be between10-30 nucleotides in length. The spacer extension sequence can bebetween 30-70 nucleotides in length.

The spacer extension sequence can comprise another moiety (e.g., astability control sequence, an endoribonuclease binding sequence, aribozyme). The moiety can decrease or increase the stability of anucleic acid targeting nucleic acid. The moiety can be a transcriptionalterminator segment (i.e., a transcription termination sequence). Themoiety can function in a eukaryotic cell. The moiety can function in aprokaryotic cell. The moiety can function in both eukaryotic andprokaryotic cells. Non-limiting examples of suitable moieties include: a5′ cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence(e.g., to allow for regulated stability and/or regulated accessibilityby proteins and protein complexes), a sequence that forms a dsRNA duplex(i.e., a hairpin), a sequence that targets the RNA to a subcellularlocation (e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

Spacer Sequence

The spacer sequence hybridizes to a sequence in a target nucleic acid ofinterest. The spacer of a genome-targeting nucleic acid can interactwith a target nucleic acid in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest.

In a CRISPR/Cas system herein, the spacer sequence can be designed tohybridize to a target nucleic acid that is located 5′ of a PAM of theCas9 enzyme used in the system. The spacer can perfectly match thetarget sequence or can have mismatches. Each Cas9 enzyme has aparticular PAM sequence that it recognizes in a target DNA. For example,S. pyogenes recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence.

The target nucleic acid sequence can comprise 20 nucleotides. The targetnucleic acid can comprise less than 20 nucleotides. The target nucleicacid can comprise more than 20 nucleotides. The target nucleic acid cancomprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. In some examples, the target nucleic acid comprisesat most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or morenucleotides. The target nucleic acid sequence can comprise 20 basesimmediately 5′ of the first nucleotide of the PAM. For example, in asequence comprising 5′-NNNNNNNNNNNNNNNNNNNNNRG-3′ (SEQ ID NO. 1,410,473)the target nucleic acid can comprise the sequence that corresponds tothe Ns, wherein N is any nucleotide, and the underlined NRG sequence isthe S. pyogenes PAM.

The spacer sequence that hybridizes to the target nucleic acid can havea length of at least about 6 nucleotides (nt). The spacer sequence canbe at least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer sequence cancomprise 20 nucleotides. The spacer sequence can comprise 19nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5′-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which can bethought of as a bulge or bulges.

The spacer sequence can be designed or chosen using a computer program.The computer program can use variables, such as predicted meltingtemperature, secondary structure formation, predicted annealingtemperature, sequence identity, genomic context, chromatinaccessibility, % GC, frequency of genomic occurrence (e.g., of sequencesthat are identical or are similar but vary in one or more spots as aresult of mismatch, insertion or deletion), methylation status, presenceof SNPs, and the like.

Minimum CRISPR Repeat Sequence

A minimum CRISPR repeat sequence is a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

A minimum CRISPR repeat sequence can comprise nucleotides that canhybridize to a minimum tracrRNA sequence in a cell. The minimum CRISPRrepeat sequence and a minimum tracrRNA sequence can form a duplex, i.e.a base-paired double-stranded structure. Together, the minimum CRISPRrepeat sequence and the minimum tracrRNA sequence can bind to thesite-directed polypeptide. At least a part of the minimum CRISPR repeatsequence can hybridize to the minimum tracrRNA sequence. At least a partof the minimum CRISPR repeat sequence can comprise at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% complementary to theminimum tracrRNA sequence. At least a part of the minimum CRISPR repeatsequence can comprise at most about 30%, about 40%, about 50%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or 100% complementary to the minimum tracrRNA sequence.

The minimum CRISPR repeat sequence can have a length from about 7nucleotides to about 100 nucleotides. For example, the length of theminimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt,from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, fromabout 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, orfrom about 15 nt to about 25 nt. In some examples, the minimum CRISPRrepeat sequence is approximately 9 nucleotides in length. The minimumCRISPR repeat sequence can be approximately 12 nucleotides in length.

The minimum CRISPR repeat sequence can be at least about 60% identicalto a reference minimum CRISPR repeat sequence (e.g., wild-type crRNAfrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the minimum CRISPR repeat sequence is at leastabout 65% identical, at least about 70% identical, at least about 75%identical, at least about 80% identical, at least about 85% identical,at least about 90% identical, at least about 95% identical, at leastabout 98% identical, at least about 99% identical or 100% identical to areference minimum CRISPR repeat sequence over a stretch of at least 6,7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

A minimum tracrRNA sequence can be a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).

A minimum tracrRNA sequence can comprise nucleotides that hybridize to aminimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequenceand a minimum CRISPR repeat sequence form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat bind to a site-directed polypeptide. At leasta part of the minimum tracrRNA sequence can hybridize to the minimumCRISPR repeat sequence. The minimum tracrRNA sequence can be at leastabout 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or 100% complementaryto the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

Bulges

In some cases, there can be a “bulge” in the duplex between the minimumCRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region ofnucleotides within the duplex. A bulge can contribute to the binding ofthe duplex to the site-directed polypeptide. The bulge can comprise, onone side of the duplex, an unpaired 5′-XXXY-3′ where X is any purine andY comprises a nucleotide that can form a wobble pair with a nucleotideon the opposite strand, and an unpaired nucleotide region on the otherside of the duplex. The number of unpaired nucleotides on the two sidesof the duplex can be different.

In one example, the bulge can comprise an unpaired purine (e.g.,adenine) on the minimum CRISPR repeat strand of the bulge. In someexamples, the bulge can comprise an unpaired 5′-AAGY-3′ of the minimumtracrRNA sequence strand of the bulge, where Y comprises a nucleotidethat can form a wobble pairing with a nucleotide on the minimum CRISPRrepeat strand.

A bulge on the minimum CRISPR repeat side of the duplex can comprise atleast 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on theminimum CRISPR repeat side of the duplex can comprise at most 1, 2, 3,4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPRrepeat side of the duplex can comprise 1 unpaired nucleotide.

A bulge on the minimum tracrRNA sequence side of the duplex can compriseat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides.A bulge on the minimum tracrRNA sequence side of the duplex can compriseat most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. Abulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) can comprise 4 unpaired nucleotides.

A bulge can comprise at least one wobble pairing. In some examples, abulge comprises at most one wobble pairing. In some examples, a bulgecan comprise at least one purine nucleotide. A bulge can comprise atleast 3 purine nucleotides. A bulge sequence can comprise at least 5purine nucleotides. A bulge sequence can comprise at least one guaninenucleotide. A bulge sequence can comprise at least one adeninenucleotide.

Hairpins

In various examples, one or more hairpins can be located 3′ to theminimum tracrRNA in the 3′ tracrRNA sequence.

The hairpin can start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 or more nucleotides 3′ from the last paired nucleotide in theminimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpincan start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides 3′ of the last paired nucleotide in the minimum CRISPRrepeat and minimum tracrRNA sequence duplex.

The hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 20 or more consecutive nucleotides. The hairpin can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutivenucleotides.

The hairpin can comprise a CC dinucleotide (i.e., two consecutivecytosine nucleotides).

The hairpin can comprise duplexed nucleotides (e.g., nucleotides in ahairpin, hybridized together). For example, a hairpin can comprise a CCdinucleotide that is hybridized to a GG dinucleotide in a hairpin duplexof the 3′ tracrRNA sequence.

One or more of the hairpins can interact with guide RNA-interactingregions of a site-directed polypeptide.

In some examples, there are two or more hairpins, and in other examplesthere are three or more hairpins.

3′ tracrRNA Sequence

A 3′ tracrRNA sequence can comprise a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

The 3′ tracrRNA sequence can have a length from about 6 nucleotides toabout 100 nucleotides. For example, the 3′ tracrRNA sequence can have alength from about 6 nucleotides (nt) to about 50 nt, from about 6 nt toabout 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The3′ tracrRNA sequence can have a length of approximately 14 nucleotides.

The 3′ tracrRNA sequence can be at least about 60% identical to areference 3′ tracrRNA sequence (e.g., wild type 3′ tracrRNA sequencefrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the 3′ tracrRNA sequence can be at least about60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, about 95% identical, about 98% identical, about 99%identical, or 100% identical, to a reference 3′ tracrRNA sequence (e.g.,wild type 3′ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

The 3′ tracrRNA sequence can comprise more than one duplexed region(e.g., hairpin, hybridized region). The 3′ tracrRNA sequence cancomprise two duplexed regions.

The 3′ tracrRNA sequence can comprise a stem loop structure. The stemloop structure in the 3′ tracrRNA can comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem loop structure inthe 3′ tracrRNA can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore nucleotides. The stem loop structure can comprise a functionalmoiety. For example, the stem loop structure can comprise an aptamer, aribozyme, a protein-interacting hairpin, a CRISPR array, an intron, oran exon. The stem loop structure can comprise at least about 1, 2, 3, 4,or 5 or more functional moieties. The stem loop structure can compriseat most about 1, 2, 3, 4, or 5 or more functional moieties.

The hairpin in the 3′ tracrRNA sequence can comprise a P-domain. In someexamples, the P-domain can comprise a double-stranded region in thehairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence can be provided whether the tracrRNA is inthe context of single-molecule guides or double-molecule guides. ThetracrRNA extension sequence can have a length from about 1 nucleotide toabout 400 nucleotides. The tracrRNA extension sequence can have a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, or 400 nucleotides. The tracrRNA extension sequence can have alength from about 20 to about 5000 or more nucleotides. The tracrRNAextension sequence can have a length of more than 1000 nucleotides. ThetracrRNA extension sequence can have a length of less than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 1000 nucleotides. The tracrRNA extension sequence can comprise lessthan 10 nucleotides in length. The tracrRNA extension sequence can be10-30 nucleotides in length. The tracrRNA extension sequence can be30-70 nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt. Thefunctional moiety can function in a eukaryotic cell. The functionalmoiety can function in a prokaryotic cell. The functional moiety canfunction in both eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moietiesinclude a 3′ poly-adenylated tail, a riboswitch sequence (e.g., to allowfor regulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like). The tracrRNA extension sequence cancomprise a primer binding site or a molecular index (e.g., barcodesequence). The tracrRNA extension sequence can comprise one or moreaffinity tags.

Single-Molecule Guide Linker Sequence

The linker sequence of a single-molecule guide nucleic acid can have alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused, Science, 337(6096):816-821 (2012). An illustrative linker has alength from about 3 nucleotides (nt) to about 90 nt, from about 3 nt toabout 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, fromabout 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3nt to about 10 nt. For example, the linker can have a length from about3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt toabout 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt,from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, fromabout 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90nt to about 100 nt. The linker of a single-molecule guide nucleic acidcan be between 4 and 40 nucleotides. The linker can be at least about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides. The linker can be at most about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used, Science, 337(6096):816-821 (2012), butnumerous other sequences, including longer sequences can likewise beused.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can one or more features, including an aptamer, aribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

Genome Engineering Strategies to Correct Cells by Deletion (Excision),Insertion, or Replacement (Deletion and Insertion) of One or More Exonsor Aberrant Intronic Splice Acceptor or Donor Sites

A step of the ex vivo methods of the present disclosure involvesediting/correcting the DMD patient specific iPS cells using genomeengineering. Likewise, a step of the in vivo methods of the presentdisclosure involves editing/correcting the muscle cells in a DMD patientusing genome engineering. Similarly, a step in the cellular methods ofthe present disclosure involves editing/correcting the dystrophin genein a human cell by genome engineering.

DMD patients exhibit a wide range of mutations in the dystrophin gene.Therefore, different patients will generally require differentcorrection strategies. Any CRISPR endonuclease can be used in themethods of the present disclosure, each CRISPR endonuclease having itsown associated PAM, which may or may not be disease specific. Forexample, gRNA spacer sequences for targeting the dystrophin gene with aCRISPR/Cas9 endonuclease from S. pyogenes have been identified in SEQ IDNOs: 1-467,030 and 1,410,430-1,410,472 of the Sequence Listing. gRNAspacer sequences for targeting the dystrophin gene with a CRISPR/Cas9endonuclease from S. aureus have been identified in SEQ ID NOs:467,031-528,196 of the Sequence Listing. gRNA spacer sequences fortargeting the dystrophin gene with a CRISPR/Cas9 endonuclease from S.thermophilus have been identified in SEQ ID NOs: 528,197-553,198 of theSequence Listing. gRNA spacer sequences for targeting the dystrophingene with a CRISPR/Cas9 endonuclease from T. denticola have beenidentified in SEQ ID NOs: 553,199-563,911 of the Sequence Listing. gRNAspacer sequences for targeting the dystrophin gene with a CRISPR/Cas9endonuclease from N. meningitides have been identified in SEQ ID NOs:563,912-627,854 and 1,410,400-1,410,402 of the Sequence Listing. gRNAspacer sequences for targeting the dystrophin gene with a CRISPR/Cpf1endonuclease from Acidominoccoccus, Lachnospiraceae and FranciscellaNovicida have been identified in SEQ ID NOs: 627,855-1,410,399 and1,410,403-1,410,429 of the Sequence Listing.

One genome engineering strategy involves exon deletion. Targeteddeletion of specific exons can be an attractive strategy for treating alarge subset of patients with a single therapeutic cocktail. It ispredicted that single exon deletions can treat up to 13% of patients,while a multi-exon deletion can treat up to 62% of patients by restoringthe dystrophin reading frame. While multi-exon deletions can reach alarger number of patients, for larger deletions the efficiency ofdeletion greatly decreases with increased size. Therefore, preferreddeletions can range from 400 to 350,000 base pairs (bp) in size. Forexample, deletions can range from 400-1,000; 1,000-5,000; 5,000-10,000,10,000-25,000; 25,000-50,000, 50,000-100,000; 100,000-200,000; or200,000-350,000 base pairs in size.

As stated previously, the DMD gene contains 79 exons. Any one or more ofthe 79 exons, or aberrant intronic splice acceptor or donor sites, canbe deleted in order to restore the dystrophin reading frame. The methodsprovide gRNA pairs that can be used to delete exons 2, 8, 43, 44, 45,46, 50, 51, 52, 53, 70, 45-53, or 45-55, as these are the regions thatare predicted to reach the largest subset of patients (see Tables 1 and2; Table 2 percentages given are the average reported from theliterature).

Different regions of the DMD gene can be repaired by either deletionand/or HDR. Certain combinations of gRNAs that cut within the genomicregion of interest can be used to correct mutations in the targetedexon. Coordinates are based on the GRch38/hg38 genomic assembly (Table1).

TABLE 1 Targeted Exon(s) Repair strategy Genomic Coordinates 45-55Deletion and/or HDR Chrx:31512453-32216916 45-53 Deletion and/or HDRChrx:31679586-32216916  2 Deletion and/or HDR Chrx:32849820-33211282  8Deletion and/or HDR Chrx:32697998-32809493 43 Deletion and/or HDRChrx:32217063-32310082 44 Deletion and/or HDR Chrx:31968514-32287529 45Deletion and/or HDR Chrx:31932227-32216916 46 Deletion and/or HDRChrx:31929745-31968339 50 Deletion and/or HDR Chrx:31774192-31836718 51Deletion and/or HDR Chrx:31729748-31819975 52 Deletion and/or HDRChrx:31679586-31773960 53 Deletion and/or HDR Chrx:31658144-31729631 70HDR Chrx:31177970-31180370

TABLE 2 Deleted % of Exon(s) Mutations Citation 45-55 62.1 Beroud, C.,et al., Hum Mutat, 2007. 28(2): p. 196-202. 45-53 53.3 Tuffery-Giraud,S., et al., Hum Mutat, 2009. 30(6): p. 934-45.  2 1.9 Aartsma-Rus, A.,et al., Hum Mutat, 2009. 30(3): p. 293-9.  8 2.2 Aartsma-Rus, A., etal., Id. Bladen, C.L., et al., Hum Mutat, 2015. 36(4): p. 395-402. 435.7 Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id. 44 6.7Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id. 45 8.6Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id. 46 4.5Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id. 50 3.9Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id. 51 13.5Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id. 52 3.9Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id. 53 8.9Aartsma-Rus, A., et al., Id. Bladen, C.L., et al., Id.

The methods provide gRNA pairs that delete exon 2 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 2 and the other gRNAcutting at the 3′ end of exon 2.

The methods provide gRNA pairs that delete exon 8 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 8 and the other gRNAcutting at the 3′ end of exon 8.

The methods provide gRNA pairs that delete exon 43 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 43 and the other gRNAcutting at the 3′ end of exon 43.

The methods provide gRNA pairs that delete exon 44 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 44 and the other gRNAcutting at the 3′ end of exon 44.

The methods provide gRNA pairs that delete exon 45 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 45 and the other gRNAcutting at the 3′ end of exon 45.

The methods provide gRNA pairs that delete exon 46 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 46 and the other gRNAcutting at the 3′ end of exon 46.

The methods provide gRNA pairs that delete exon 50 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 50 and the other gRNAcutting at the 3′ end of exon 50.

The methods provide gRNA pairs that delete exon 51 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 51 and the other gRNAcutting at the 3′ end of exon 51.

The methods provide gRNA pairs that delete exon 52 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 52 and the other gRNAcutting at the 3′ end of exon 52.

The methods provide gRNA pairs that delete exon 53 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 53 and the other gRNAcutting at the 3′ end of exon 53.

The methods provide gRNA pairs that delete exon 70 by cutting the genetwice, one gRNA cutting at the 5′ end of exon 70 and the other gRNAcutting at the 3′ end of exon 70.

The methods provide gRNA pairs that delete exons 45-53 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 45 and the other gRNAcutting at the 3′ end of exon 53.

The methods provide gRNA pairs that delete exons 45-55 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 45 and the other gRNAcutting at the 3′ end of exon 55.

Another genome engineering strategy involves insertion or replacement ofone or more exons or aberrant intronic splice acceptor or donor sites byhomology directed repair (HDR), which is also known as homologousrecombination (HR). Homology directed repair is one strategy fortreating patients that have premature stop codons due to smallinsertions/deletions or point mutations. Rather than making a largegenomic deletion that will convert a DMD phenotype to a BMD phenotype,this strategy will restore the entire reading frame and completelyreverse the diseased state. This strategy will require a more customapproach based on the location of the patient's pre-mature stop. Most ofthe dystrophin exons are small (<300 bp). This is advantageous, as HDRefficiencies are inversely related to the size of the donor molecule.Also, it is expected that the donor templates can fit into sizeconstrained adeno-associated virus (AAV) molecules, which have beenshown to be an effective means of donor template delivery.

Homology direct repair is a cellular mechanism for repairingdouble-stranded breaks (DSBs). The most common form is homologousrecombination. There are additional pathways for HDR, includingsingle-strand annealing and alternative-HDR. Genome engineering toolsallow researchers to manipulate the cellular homologous recombinationpathways to create site-specific modifications to the genome. It hasbeen found that cells can repair a double-stranded break using asynthetic donor molecule provided in trans. Therefore, by introducing adouble-stranded break near a specific mutation and providing a suitabledonor, targeted changes can be made in the genome. Specific cleavageincreases the rate of HDR more than 1,000 fold above the rate of 1 in10⁶ cells receiving a homologous donor alone. The rate of homologydirected repair (HDR) at a particular nucleotide is a function of thedistance to the cut site, so choosing overlapping or nearest targetsites is important. Gene editing offers the advantage over geneaddition, as correcting in situ leaves the rest of the genomeunperturbed.

Supplied donors for editing by HDR vary markedly but can contain theintended sequence with small or large flanking homology arms to allowannealing to the genomic DNA. The homology regions flanking theintroduced genetic changes can be 30 bp or smaller, or as large as amulti-kilobase cassette that can contain promoters, cDNAs, etc. Bothsingle-stranded and double-stranded oligonucleotide donors have beenused. These oligonucleotides can range in size from less than 100 nt toover 200 nt, though longer ssDNA can also be generated and used.Double-stranded donors can be used, including PCR amplicons, plasmids,and mini-circles. In general, it has been found that an AAV vector canbe a very effective means of delivery of a donor template, though thepackaging limits for individual donors is <5 kb. Active transcription ofthe donor increased HDR three-fold, indicating the inclusion of promotercan increase conversion. Conversely, CpG methylation of the donordecreased gene expression and HDR.

In addition to wildtype endonucleases, such as Cas9, nickase variantsexist that can have one or the other nuclease domain inactivatedresulting in cutting of only one DNA strand. HDR can be directed fromindividual Cas nickases or using pairs of nickases that flank the targetarea. Donors can be single-stranded, nicked, or dsDNA.

The donor DNA can be supplied with the nuclease or independently by avariety of different methods, for example by transfection,nano-particle, micro-injection, or viral transduction. A range oftethering options has been proposed to increase the availability of thedonors for HDR. Examples include attaching the donor to the nuclease,attaching to DNA binding proteins that bind nearby, or attaching toproteins that are involved in DNA end binding or repair.

The repair pathway choice can be guided by a number of cultureconditions, such as those that influence cell cycling, or by targetingof DNA repair and associated proteins. For example, to increase HDR, keyNHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

Without a donor present, the ends from a DNA break or ends fromdifferent breaks can be joined using the several nonhomologous repairpathways in which the DNA ends are joined with little or no base-pairingat the junction. In addition to canonical NHEJ, there are similar repairmechanisms, such as alt-NHEJ. If there are two breaks, the interveningsegment can be deleted or inverted. NHEJ repair pathways can lead toinsertions, deletions, or mutations at the joints.

NHEJ was used to insert a 15-kb inducible gene expression cassette intoa defined locus in human cell lines after nuclease cleavage. Maresca,M., Lin, V. G., Guo, N. & Yang, Y. Obligate ligation-gated recombination(ObLiGaRe): custom-designed nuclease-mediated targeted integrationthrough nonhomologous end joining. Genome Res 23, 539-546 (2013).

In addition to genome editing by NHEJ or HDR, site-specific geneinsertions have been conducted that use both the NHEJ pathway and HR. Acombination approach can be applicable in certain settings, possiblyincluding intron/exon borders. NHEJ can prove effective for ligation inthe intron, while the error-free HDR can be better suited in the codingregion.

As stated previously, the DMD gene contains 79 exons. Any one or more ofthe 79 exons can be repaired in order to correct a mutation and restorethe dystrophin reading frame. Some methods provide one gRNA or a pair ofgRNAs that can be used to facilitate incorporation of a new sequencefrom a polynucleotide donor template to insert or replace a sequence inexon 70, as data shows that exon 70 can be prone to the most prematurestop codons in the dystrophin gene (Tuffery-Giraud, S., et al., HumMutat, 2009. 30(6): p. 934-45) (Flanigan, K. M., et al., Hum Mutat,2009. 30(12): p. 1657-66). In order to make the method applicable to thelargest number of patients, the method involves a donor template thatcan insert or replace the whole exon 70. Alternatively, the methodsprovide one gRNA or a pair of gRNAs that can be used to facilitateincorporation of a new sequence from a polynucleotide donor template toinsert or replace a sequence in exon 2, exon 8, exon 43, exon 44, exon45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. See Table1.

In order to ensure that the pre-mRNA is properly processed followingHDR, it is important to keep the surrounding splicing signals intact.Splicing donor and acceptors can be generally within 100 base pairs ofthe neighboring intron. Therefore, in some examples, methods can provideall gRNAs that cut approximately +/−0-3100 bp with respect to the exon'sintron junctions.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 2 and the other gRNAcutting at the 3′ end of exon 2 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 2.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 2.

Some examples of the methods provide gRNA pairs that make a deletion bycutting the gene twice, one gRNA cutting at the 5′ end of exon 8 and theother gRNA cutting at the 3′ end of exon 8 that facilitatesincorporation of a new sequence from a polynucleotide donor template toreplace a sequence in exon 8.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 8.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 43 and the other gRNAcutting at the 3′ end of exon 43 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 43.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 43.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 44 and the other gRNAcutting at the 3′ end of exon 44 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 44.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 44.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 45 and the other gRNAcutting at the 3′ end of exon 45 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 45.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 45.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 46 and the other gRNAcutting at the 3′ end of exon 46 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 46.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 46.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 50 and the other gRNAcutting at the 3′ end of exon 50 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 50.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 50.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 51 and the other gRNAcutting at the 3′ end of exon 51 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 51.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 51.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 52 and the other gRNAcutting at the 3′ end of exon 52 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 52.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 52.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 53 and the other gRNAcutting at the 3′ end of exon 53 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 53.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 53.

Some methods provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of exon 70 and the other gRNAcutting at the 3′ end of exon 70 that facilitates incorporation of a newsequence from a polynucleotide donor template to replace a sequence inexon 70.

Alternatively, some methods provide one gRNA from the precedingparagraph to make one double-strand cut that facilitates insertion of anew sequence from a polynucleotide donor template to replace a sequencein exon 70.

In addition to single exon replacements by homology directed repair, wealso describe methods for conducting a partial cDNA knock-in ofmutational hotspots found in the DMD gene. For example, a treatment thatrepairs exons 45-55 can treat up to 62% of patients. Rather thandeleting or replacing exons 45-55 as described herein, another treatmentoption replaces entire genomic region for exons 45-55—which, includingintrons, spans >350,000 bp—with a cDNA containing only the coding regionof exons 45-55, which spans approximate 1800 bp. The replacement couldbe effected using a homology directed repair approach. By excluding theintergenic regions, the cDNA for exons 45-55 can more easily beaccommodated (than the entire genomic region) along with homology armsinto any donor vector described in the section of this applicationtitled Nucleic Acids Encoding System Components. In this approach, twogRNAs and Cas9 or Cpf1 that remove the genomic region from exon 45-55can be delivered along with a donor construct to replace the deletedregion with the desired cDNA knock-in.

The cDNA knock-in approach can be used to replace any series of exons.

The cDNA knock-in sequence can be optimized to contain synthetic intronsequences. Synthetic introns which are smaller than naturally occurringintrons can be added between the exons in the donor construct to ensureproper expression and processing of the DMD locus.

Illustrative modifications within the dystrophin gene include deletions,insertions, or replacements within or proximal to the dystrophin locireferred to above, such as within the region of less than 3 kb, lessthan 2 kb, less than 1 kb, less than 0.5 kb upstream or downstream ofthe specific exon. Given the relatively wide variations of mutations inthe dystrophin gene, it will be appreciated that numerous variations ofthe deletions, insertions, or replacements referenced above (includingwithout limitation larger as well as smaller deletions), would beexpected to result in restoration of the dystrophin reading frame andrestoration of the dystrophin protein activity.

Such variants can include deletions, insertions, or replacements thatare larger in the 5′ and/or 3′ direction than the specific exon inquestion, or smaller in either direction. Accordingly, by “near” or“proximal” with respect to specific exon deletions, insertions orreplacements, it is intended that the SSB or DSB locus associated with adesired deletion, insertion, or replacement boundary (also referred toherein as an endpoint) can be within a region that is less than about 3kb from the reference locus noted. The SSB or DSB locus can be moreproximal and within 2 kb, within 1 kb, within 0.5 kb, or within 0.1 kb.In the case of small deletions, the desired endpoint can be at or“adjacent to” the reference locus, by which it is intended that theendpoint can be within 100 bp, within 50 bp, within 25 bp, or less thanabout 10 bp to 5 bp from the reference locus.

One advantage for patients with DMD of replicating or mimicking theproduct produced by exon skipping and/or restoring the reading frame isthat it is already known to be both safe and associated with theamelioration of DMD. Other examples comprising larger or smallerdeletions/insertions/replacements can be expected to provide the samebenefit, as long as the dystrophin reading frame is restored. Thus, itcan be expected that many variations of the deletions, insertions, andreplacements described and illustrated herein can be effective forameliorating DMD.

Target Sequence Selection

Shifts in the location of the 5′ boundary and/or the 3′ boundaryrelative to particular reference loci can be used to facilitate orenhance particular applications of gene editing, which depend in part onthe endonuclease system selected for the editing, as further describedand illustrated herein.

In a first nonlimiting example of such target sequence selection, manyendonuclease systems have rules or criteria that can guide the initialselection of potential target sites for cleavage, such as therequirement of a PAM sequence motif in a particular position adjacent tothe DNA cleavage sites in the case of CRISPR Type II or Type Vendonucleases.

In another nonlimiting example of target sequence selection oroptimization, the frequency of “off-target” activity for a particularcombination of target sequence and gene editing endonuclease (i.e. thefrequency of DSBs occurring at sites other than the selected targetsequence) can be assessed relative to the frequency of on-targetactivity. In some cases, cells that have been correctly edited at thedesired locus can have a selective advantage relative to other cells.Illustrative, but nonlimiting, examples of a selective advantage includethe acquisition of attributes such as enhanced rates of replication,persistence, resistance to certain conditions, enhanced rates ofsuccessful engraftment or persistence in vivo following introductioninto a patient, and other attributes associated with the maintenance orincreased numbers or viability of such cells. In other cases, cells thathave been correctly edited at the desired locus can be positivelyselected for by one or more screening methods used to identify, sort orotherwise select for cells that have been correctly edited. Bothselective advantage and directed selection methods can take advantage ofthe phenotype associated with the correction. In some cases, cells canbe edited two or more times in order to create a second modificationthat creates a new phenotype that is used to select or purify theintended population of cells. Such a second modification could becreated by adding a second gRNA for a selectable or screenable marker.In some cases, cells can be correctly edited at the desired locus usinga DNA fragment that contains the cDNA and also a selectable marker.

Whether any selective advantage is applicable or any directed selectionis to be applied in a particular case, target sequence selection canalso be guided by consideration of off-target frequencies in order toenhance the effectiveness of the application and/or reduce the potentialfor undesired alterations at sites other than the desired target. Asdescribed further and illustrated herein and in the art, the occurrenceof off-target activity can be influenced by a number of factorsincluding similarities and dissimilarities between the target site andvarious off target sites, as well as the particular endonuclease used.Bioinformatics tools are available that assist in the prediction ofoff-target activity, and frequently such tools can also be used toidentify the most likely sites of off-target activity, which can then beassessed in experimental settings to evaluate relative frequencies ofoff-target to on-target activity, thereby allowing the selection ofsequences that have higher relative on-target activities. Illustrativeexamples of such techniques are provided herein, and others are known inthe art.

Another aspect of target sequence selection relates to homologousrecombination events. Sequences sharing regions of homology can serve asfocal points for homologous recombination events that result in deletionof intervening sequences. Such recombination events occur during thenormal course of replication of chromosomes and other DNA sequences, andalso at other times when DNA sequences are being synthesized, such as inthe case of repairs of double-strand breaks (DSBs), which occur on aregular basis during the normal cell replication cycle but can also beenhanced by the occurrence of various events (such as UV light and otherinducers of DNA breakage) or the presence of certain agents (such asvarious chemical inducers). Many such inducers cause DSBs to occurindiscriminately in the genome, and DSBs can be regularly induced andrepaired in normal cells. During repair, the original sequence can bereconstructed with complete fidelity, however, in some cases, smallinsertions or deletions (referred to as “indels”) are introduced at theDSB site.

DSBs can also be specifically induced at particular locations, as in thecase of the endonucleases systems described herein, which can be used tocause directed or preferential gene modification events at selectedchromosomal locations. The tendency for homologous sequences to besubject to recombination in the context of DNA repair (as well asreplication) can be taken advantage of in a number of circumstances, andis the basis for one application of gene editing systems, such asCRISPR, in which homology directed repair is used to insert a sequenceof interest, provided through use of a “donor” polynucleotide, into adesired chromosomal location.

Regions of homology between particular sequences, which can be smallregions of “microhomology” that can comprise as few as ten basepairs orless, can also be used to bring about desired deletions. For example, asingle DSB can be introduced at a site that exhibits microhomology witha nearby sequence. During the normal course of repair of such DSB, aresult that occurs with high frequency is the deletion of theintervening sequence as a result of recombination being facilitated bythe DSB and concomitant cellular repair process.

In some circumstances, however, selecting target sequences withinregions of homology can also give rise to much larger deletions,including gene fusions (when the deletions are in coding regions), whichmay or may not be desired given the particular circumstances.

The examples provided herein further illustrate the selection of varioustarget regions for the creation of DSBs designed to induce disruptions,deletions, or replacements that result in restoration of the dystrophinreading frame, as well as the selection of specific target sequenceswithin such regions that are designed to minimize off-target eventsrelative to on-target events.

Nucleic Acid Modifications

In some cases, polynucleotides introduced into cells can comprise one ormore modifications that can be used, individually or in combination, forexample, to enhance activity, stability or specificity, alter delivery,reduce innate immune responses in host cells, or for other enhancements,as further described herein and known in the art.

In certain examples, modified polynucleotides can be used in theCRISPR/Cas9/Cpf1 system, in which case the guide RNAs (eithersingle-molecule guides or double-molecule guides) and/or a DNA or an RNAencoding a Cas or Cpf1 endonuclease introduced into a cell can bemodified, as described and illustrated below. Such modifiedpolynucleotides can be used in the CRISPR/Cas9/Cpf1 system to edit anyone or more genomic loci.

Using the CRISPR/Cas9/Cpf1 system for purposes of nonlimitingillustrations of such uses, modifications of guide RNAs can be used toenhance the formation or stability of the CRISPR/Cas9/Cpf1 genomeediting complex comprising guide RNAs, which can be single-moleculeguides or double-molecule, and a Cas or Cpf1 endonuclease. Modificationsof guide RNAs can also or alternatively be used to enhance theinitiation, stability or kinetics of interactions between the genomeediting complex with the target sequence in the genome, which can beused, for example, to enhance on-target activity. Modifications of guideRNAs can also or alternatively be used to enhance specificity, e.g., therelative rates of genome editing at the on-target site as compared toeffects at other (off-target) sites.

Modifications can also or alternatively be used to increase thestability of a guide RNA, e.g., by increasing its resistance todegradation by ribonucleases (RNases) present in a cell, thereby causingits half-life in the cell to be increased. Modifications enhancing guideRNA half-life can be particularly useful in aspects in which a Cas orCpf1 endonuclease is introduced into the cell to be edited via an RNAthat needs to be translated in order to generate endonuclease, becauseincreasing the half-life of guide RNAs introduced at the same time asthe RNA encoding the endonuclease can be used to increase the time thatthe guide RNAs and the encoded Cas or Cpf1 endonuclease co-exist in thecell.

Modifications can also or alternatively be used to decrease thelikelihood or degree to which RNAs introduced into cells elicit innateimmune responses. Such responses, which have been well characterized inthe context of RNA interference (RNAi), including small-interfering RNAs(siRNAs), as described below and in the art, tend to be associated withreduced half-life of the RNA and/or the elicitation of cytokines orother factors associated with immune responses.

One or more types of modifications can also be made to RNAs encoding anendonuclease that are introduced into a cell, including, withoutlimitation, modifications that enhance the stability of the RNA (such asby increasing its degradation by RNAses present in the cell),modifications that enhance translation of the resulting product (i.e.the endonuclease), and/or modifications that decrease the likelihood ordegree to which the RNAs introduced into cells elicit innate immuneresponses.

Combinations of modifications, such as the foregoing and others, canlikewise be used. In the case of CRISPR/Cas9/Cpf1, for example, one ormore types of modifications can be made to guide RNAs (including thoseexemplified above), and/or one or more types of modifications can bemade to RNAs encoding Cas endonuclease (including those exemplifiedabove).

By way of illustration, guide RNAs used in the CRISPR/Cas9/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means,enabling a number of modifications to be readily incorporated, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach that can be used for generatingchemically-modified RNAs of greater length is to produce two or moremolecules that are ligated together. Much longer RNAs, such as thoseencoding a Cas9 endonuclease, are more readily generated enzymatically.While fewer types of modifications are available for use inenzymatically produced RNAs, there are still modifications that can beused to, e.g., enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as describedfurther below and in the art; and new types of modifications areregularly being developed.

By way of illustration of various types of modifications, especiallythose used frequently with smaller chemically synthesized RNAs,modifications can comprise one or more nucleotides modified at the 2′position of the sugar, in some aspects a 2′-O-alkyl, 2′-O-alkyl-O-alkyl,or 2′-fluoro-modified nucleotide. In some aspects, RNA modifications cancomprise 2′-fluoro, 2′-amino or 2′ O-methyl modifications on the riboseof pyrimidines, abasic residues, or an inverted base at the 3′ end ofthe RNA. Such modifications can be routinely incorporated intooligonucleotides and these oligonucleotides have been shown to have ahigher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligonucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Some oligonucleotides are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH, ˜N(CH3)˜O˜CH2 (known as amethylene(methylimino) or MMI backbone), CH2-O—N(CH3)-CH2,CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones[see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)];morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch and DavidCorey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al.,Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci.,97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3 OCH3,OCH3 O(CH2)n CH3, O(CH2)n NH2, or O(CH2)n CH3, where n is from 1 toabout 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; CI; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O—, S-,or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. In some aspects, amodification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as2′-O-(2-methoxyethyl)) (Martin et al, Helv. Chim. Acta, 1995, 78, 486).Other modifications include 2′-methoxy (2′-O—CH3), 2′-propoxy (2′-OCH2CH2CH3) and 2′-fluoro (2′-F). Similar modifications can also be made atother positions on the oligonucleotide, particularly the 3′ position ofthe sugar on the 3′ terminal nucleotide and the 5′ position of 5′terminal nucleotide. Oligonucleotides can also have sugar mimetics, suchas cyclobutyls in place of the pentofuranosyl group.

In some examples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups. Thebase units can be maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can bereplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases can be retained and bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds comprise, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al, Science, 254: 1497-1500 (1991).

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, pp 75-77 (1980); Gebeyehu et al., Nucl. Acids Res.15:4513 (1997). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke,S. T. and Lebleu, B., eds., Antisense Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278) and are aspects of basesubstitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Further, nucleobases can comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications',pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspectsof base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. Nos. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588;5,830,653; 6,005,096; and US Patent Application Publication2003/0158403.

Thus, the term “modified” refers to a non-natural sugar, phosphate, orbase that is incorporated into a guide RNA, an endonuclease, or both aguide RNA and an endonuclease. It is not necessary for all positions ina given oligonucleotide to be uniformly modified, and in fact more thanone of the aforementioned modifications can be incorporated in a singleoligonucleotide, or even in a single nucleoside within anoligonucleotide.

The guide RNAs and/or mRNA (or DNA) encoding an endonuclease can bechemically linked to one or more moieties or conjugates that enhance theactivity, cellular distribution, or cellular uptake of theoligonucleotide. Such moieties comprise, but are not limited to, lipidmoieties such as a cholesterol moiety [Letsinger et al., Proc. Natl.Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al.,Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g.,hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol orundecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) andSvinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. AcidsRes., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain[Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)];adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys.Acta, 1264: 229-237 (1995)]; or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J.Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Sugars and other moieties can be used to target proteins and complexescomprising nucleotides, such as cationic polysomes and liposomes, toparticular sites. For example, hepatic cell directed transfer can bemediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, etal., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known inthe art and regularly developed can be used to target biomolecules ofuse in the present case and/or complexes thereof to particular targetcells of interest.

These targeting moieties or conjugates can include conjugate groupscovalently bound to functional groups, such as primary or secondaryhydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisdisclosure, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860. Conjugate moieties include, but arenot limited to, lipid moieties such as a cholesterol moiety, cholicacid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, analiphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See,e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and5,688,941.

Longer polynucleotides that are less amenable to chemical synthesis andare typically produced by enzymatic synthesis can also be modified byvarious means. Such modifications can include, for example, theintroduction of certain nucleotide analogs, the incorporation ofparticular sequences or other moieties at the 5′ or 3′ ends ofmolecules, and other modifications. By way of illustration, the mRNAencoding Cas9 is approximately 4 kb in length and can be synthesized byin vitro transcription. Modifications to the mRNA can be applied to,e.g., increase its translation or stability (such as by increasing itsresistance to degradation with a cell), or to reduce the tendency of theRNA to elicit an innate immune response that is often observed in cellsfollowing introduction of exogenous RNAs, particularly longer RNAs suchas that encoding Cas9.

Numerous such modifications have been described in the art, such aspolyA tails, 5′ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) orm7G(5′)ppp(5′)G (mCAP)), modified 5′ or 3′ untranslated regions (UTRs),use of modified bases (such as Pseudo-UTP, 2-Thio-UTP,5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), ortreatment with phosphatase to remove 5′ terminal phosphates. These andother modifications are known in the art, and new modifications of RNAsare regularly being developed.

There are numerous commercial suppliers of modified RNAs, including forexample, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon andmany others. As described by TriLink, for example, 5-Methyl-CTP can beused to impart desirable characteristics, such as increased nucleasestability, increased translation or reduced interaction of innate immunereceptors with in vitro transcribed RNA.5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as wellas Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innateimmune stimulation in culture and in vivo while enhancing translation,as illustrated in publications by Kormann et al. and Warren et al.referred to below.

It has been shown that chemically modified mRNA delivered in vivo can beused to achieve improved therapeutic effects; see, e.g., Kormann et al.,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used,for example, to increase the stability of the RNA molecule and/or reduceits immunogenicity. Using chemical modifications such as Pseudo-U,N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substitutingjust one quarter of the uridine and cytidine residues with 2-Thio-U and5-Methyl-C respectively resulted in a significant decrease in toll-likereceptor (TLR) mediated recognition of the mRNA in mice. By reducing theactivation of the innate immune system, these modifications can be usedto effectively increase the stability and longevity of the mRNA in vivo;see, e.g., Kormann et al., supra.

It has also been shown that repeated administration of syntheticmessenger RNAs incorporating modifications designed to bypass innateanti-viral responses can reprogram differentiated human cells topluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs that act as primary reprogramming proteinscan be an efficient means of reprogramming multiple human cell types.Such cells are referred to as induced pluripotency stem cells (iPSCs),and it was found that enzymatically synthesized RNA incorporating5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could beused to effectively evade the cell's antiviral response; see, e.g.,Warren et al., supra.

Other modifications of polynucleotides described in the art include, forexample, the use of polyA tails, the addition of 5′ cap analogs (such asm7G(5′)ppp(5′)G (mCAP)), modifications of 5′ or 3′ untranslated regions(UTRs), or treatment with phosphatase to remove 5′ terminalphosphates—and new approaches are regularly being developed.

A number of compositions and techniques applicable to the generation ofmodified RNAs for use herein have been developed in connection with themodification of RNA interference (RNAi), including small-interferingRNAs (siRNAs). siRNAs present particular challenges in vivo becausetheir effects on gene silencing via mRNA interference are generallytransient, which can require repeat administration. In addition, siRNAsare double-stranded RNAs (dsRNA) and mammalian cells have immuneresponses that have evolved to detect and neutralize dsRNA, which isoften a by-product of viral infection. Thus, there are mammalian enzymessuch as PKR (dsRNA-responsive kinase), and potentially retinoicacid-inducible gene I (RIG-I), that can mediate cellular responses todsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) thatcan trigger the induction of cytokines in response to such molecules;see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012);Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan,Hum Gene Ther 19(2):111-24 (2008); and references cited therein.

A large variety of modifications have been developed and applied toenhance RNA stability, reduce innate immune responses, and/or achieveother benefits that can be useful in connection with the introduction ofpolynucleotides into human cells, as described herein; see, e.g., thereviews by Whitehead K A et al., Annual Review of Chemical andBiomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, MiniRev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin MolTher., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic AcidChem Chapter 16: Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19(2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsenet al., Front Genet 3:154 (2012).

As noted above, there are a number of commercial suppliers of modifiedRNAs, many of which have specialized in modifications designed toimprove the effectiveness of siRNAs. A variety of approaches are offeredbased on various findings reported in the literature. For example,Dharmacon notes that replacement of a non-bridging oxygen with sulfur(phosphorothioate, PS) has been extensively used to improve nucleaseresistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery11:125-140 (2012). Modifications of the 2′-position of the ribose havebeen reported to improve nuclease resistance of the internucleotidephosphate bond while increasing duplex stability (Tm), which has alsobeen shown to provide protection from immune activation. A combinationof moderate PS backbone modifications with small, well-tolerated2′-substitutions (2′-O-Methyl, 2′-Fluoro, 2′-Hydro) have been associatedwith highly stable siRNAs for applications in vivo, as reported bySoutschek et al. Nature 432:173-178 (2004); and 2′-O-Methylmodifications have been reported to be effective in improving stabilityas reported by Volkov, Oligonucleotides 19:191-202 (2009). With respectto decreasing the induction of innate immune responses, modifyingspecific sequences with 2′-O-Methyl, 2′-Fluoro, 2′-Hydro have beenreported to reduce TLR7/TLR8 interaction while generally preservingsilencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505(2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additionalmodifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine,5-methyluracil, and N6-methyladenosine have also been shown to minimizethe immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko,K. et al., Immunity 23:165-175 (2005).

As is also known in the art, and commercially available, a number ofconjugates can be applied to polynucleotides, such as RNAs, for useherein that can enhance their delivery and/or uptake by cells, includingfor example, cholesterol, tocopherol and folic acid, lipids, peptides,polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther.Deliv. 4:791-809 (2013), and references cited therein.

Codon-Optimization

A polynucleotide encoding a site-directed polypeptide can becodon-optimized according to methods standard in the art for expressionin the cell containing the target DNA of interest. For example, if theintended target nucleic acid is in a human cell, a human codon-optimizedpolynucleotide encoding Cas9 is contemplated for use for producing theCas9 polypeptide.

Complexes of a Genome-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A genome-targeting nucleic acid interacts with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), therebyforming a complex. The genome-targeting nucleic acid guides thesite-directed polypeptide to a target nucleic acid.

RNPs

The site-directed polypeptide and genome-targeting nucleic acid can eachbe administered separately to a cell or a patient. On the other hand,the site-directed polypeptide can be pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA. Thepre-complexed material can then be administered to a cell or a patient.Such pre-complexed material is known as a ribonucleoprotein particle(RNP).

Nucleic Acids Encoding System Components

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a genome-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, and/or any nucleic acid orproteinaceous molecule necessary to carry out the aspects of the methodsof the disclosure.

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure, a site-directed polypeptide of the disclosure, and/or anynucleic acid or proteinaceous molecule necessary to carry out theaspects of the methods of the disclosure can comprise a vector (e.g., arecombinant expression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated. Anothertype of vector is a viral vector, wherein additional nucleic acidsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some examples, vectors can be capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells, and those that direct expressionof the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the target cell, the level ofexpression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Additional vectors contemplated for eukaryotic target cells include, butare not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3, which aredescribed in FIGS. 1A to 1C. Other vectors can be used so long as theyare compatible with the host cell.

In some examples, a vector can comprise one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector. The vector can be a self-inactivatingvector that either inactivates the viral sequences or the components ofthe CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including guide RNAs used in connection withCas endonuclease, various promoters such as RNA polymerase IIIpromoters, including for example U6 and H1, can be advantageous.Descriptions of and parameters for enhancing the use of such promotersare known in art, and additional information and approaches areregularly being described; see, e.g., Ma, H. et al., MolecularTherapy—Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector can also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector can also comprise appropriate sequences for amplifyingexpression. The expression vector can also include nucleotide sequencesencoding non-native tags (e.g., histidine tag, hemagglutinin tag, greenfluorescent protein, etc.) that are fused to the site-directedpolypeptide, thus resulting in a fusion protein.

A promoter can be an inducible promoter (e.g., a heat shock promoter,tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).The promoter can be a constitutive promoter (e.g., CMV promoter, UBCpromoter). In some cases, the promoter can be a spatially restrictedand/or temporally restricted promoter (e.g., a tissue specific promoter,a cell type specific promoter, etc.).

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure and/or a site-directed polypeptide can be packaged into or onthe surface of delivery vehicles for delivery to cells. Deliveryvehicles contemplated include, but are not limited to, nanospheres,liposomes, quantum dots, nanoparticles, polyethylene glycol particles,hydrogels, and micelles. A variety of targeting moieties can be used toenhance the preferential interaction of such vehicles with desired celltypes or locations.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

Delivery

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) can be delivered by non-viral delivery vehicles known inthe art, such as electroporation or lipid nanoparticles. In furtheralternative aspects, the DNA endonuclease can be delivered as one ormore polypeptides, either alone or pre-complexed with one or more guideRNAs, or one or more crRNA together with a tracrRNA.

Polynucleotides can be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses onnon-viral delivery vehicles for siRNA that are also useful for deliveryof other polynucleotides).

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, can be delivered to a cell or a patient by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle can range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs can be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, can be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art can be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids can be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) can be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid can each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

RNA is capable of forming specific interactions with RNA or DNA. Whilethis property is exploited in many biological processes, it also comeswith the risk of promiscuous interactions in a nucleic acid-richcellular environment. One solution to this problem is the formation ofribonucleoprotein particles (RNPs), in which the RNA is pre-complexedwith an endonuclease. Another benefit of the RNP is protection of theRNA from degradation.

The endonuclease in the RNP can be modified or unmodified. Likewise, thegRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerousmodifications are known in the art and can be used.

The endonuclease and sgRNA can be combined in a 1:1 molar ratio.Alternatively, the endonuclease, crRNA and tracrRNA can be generallycombined in a 1:1:1 molar ratio. However, a wide range of molar ratioscan be used to produce a RNP.

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes can be from any AAV serotype for which recombinant viruscan be derived, and can be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12,AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in,for example, international patent application publication number WO01/83692. See Table 3.

TABLE 3 AAV Genbank Serotype Accession No. AAV-1 NC_002077.1 AAV-2NC_001401.2 AAV-3 NC_001729.1 AAV-3B AF028705.1 AAV-4 NC_001829.1 AAV-5NC_006152.1 AAV-6 AF028704.1 AAV-7 NC_006260.1 AAV-8 NC_006261.1 AAV-9AX753250.1 AAV-10 AY631965.1 AAV-11 AY631966.1 AAV-12 DQ813647.1 AAV-13EU285562.1

A method of generating a packaging cell involves creating a cell linethat stably expresses all of the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line can then be infected with ahelper virus, such as adenovirus. The advantages of this method are thatthe cells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus, rather than plasmids, to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211;5,871,982; and 6,258,595.

AAV vector serotypes can be matched to target cell types. For example,the following exemplary cell types can be transduced by the indicatedAAV serotypes among others. See Table 4.

TABLE 4 Tissue/Cell Type Serotype Liver AAV8, AAV3, AAV5, AAV9 Skeletalmuscle AAV1, AAV7, AAV6, AAV8, AAV9 Central nervous system AAV5, AAV1,AAV4 RPE AAV5, AAV4 Photoreceptor cells AAV5 Lung AAV9 Heart AAV8Pancreas AAV8 Kidney AAV2

Genetically Modified Cells

The term “genetically modified cell” refers to a cell that comprises atleast one genetic modification introduced by genome editing (e.g., usingthe CRISPR/Cas system). In some ex vivo examples herein, the geneticallymodified cell can be a genetically modified progenitor cell. In some invivo examples herein, the genetically modified cell can be a geneticallymodified muscle cell or genetically modified muscle pre-cursor cell. Agenetically modified cell comprising an exogenous genome-targetingnucleic acid and/or an exogenous nucleic acid encoding agenome-targeting nucleic acid is contemplated herein.

The term “control treated population” describes a population of cellsthat has been treated with identical media, viral induction, nucleicacid sequences, temperature, confluency, flask size, pH, etc., with theexception of the addition of the genome editing components. Any methodknown in the art can be used to measure restoration of the dystrophinreading frame, for example, Western Blot analysis of the dystrophinprotein or quantifying dystrophin mRNA.

The term “isolated cell” refers to a cell that has been removed from anorganism in which it was originally found, or a descendant of such acell. Optionally, the cell can be cultured in vitro, e.g., under definedconditions or in the presence of other cells. Optionally, the cell canbe later introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells refers to a population of cells that has been removed andseparated from a mixed or heterogeneous population of cells. In somecases, the isolated population can be a substantially pure population ofcells, as compared to the heterogeneous population from which the cellswere isolated or enriched. In some cases, the isolated population can bean isolated population of human progenitor cells, e.g., a substantiallypure population of human progenitor cells, as compared to aheterogeneous population of cells comprising human progenitor cells andcells from which the human progenitor cells were derived.

The term “substantially enhanced,” with respect to a particular cellpopulation, refers to a population of cells in which the occurrence of aparticular type of cell is increased relative to pre-existing orreference levels, by at least 2-fold, at least 3-, at least 4-, at least5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, atleast 20-, at least 50-, at least 100-, at least 400-, at least 1000-,at least 5000-, at least 20000-, at least 100000- or more folddepending, e.g., on the desired levels of such cells for amelioratingDMD.

The term “substantially enriched” with respect to a particular cellpopulation, refers to a population of cells that is at least about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or morewith respect to the cells making up a total cell population.

The term “substantially pure” with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,at least about 85%, at least about 90%, or at least about 95% pure, withrespect to the cells making up a total cell population. That is, theterms “substantially pure” or “essentially purified,” with regard to apopulation of progenitor cells, refers to a population of cells thatcontain fewer than about 20%, about 15%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, orless than 1%, of cells that are not progenitor cells as defined by theterms herein.

Differentiation of Corrected iPSCs into Pax7+ Muscle Progenitor Cells

Another step of the ex vivo methods of the present disclosure involvesdifferentiating the corrected iPSCs into Pax7+ muscle progenitor cells.The differentiating step can be performed according to any method knownin the art. For example, the differentiating step can comprisecontacting the genome-edited iPSC with specific media formulations,including small molecule drugs, to differentiate it into a Pax7+ muscleprogenitor cell, as shown in Chal, Oginuma et al. 2015. Alternatively,iPSCs, myogenic progenitors, and cells of other lineages can bedifferentiated into muscle using any one of a number of establishedmethods that involve transgene over expression, serum withdrawal, and/orsmall molecule drugs, as shown in the methods of Tapscott, Davis et al.1988, Langen, Schols et al. 2003, Fujita, Endo et al. 2010, Xu,Tabebordbar et al. 2013, Shoji, Woltjen et al. 2015.

Implanting Pax7+ Muscle Progenitor Cells into Patients

Another step of the ex vivo methods of the invention involves implantingthe Pax7+ muscle progenitor cells into patients. This implanting stepcan be accomplished using any method of implantation known in the art.For example, the genetically modified cells can be injected directly inthe patient's muscle.

Pharmaceutically Acceptable Carriers

The ex vivo methods of administering progenitor cells to a subjectcontemplated herein involve the use of therapeutic compositionscomprising progenitor cells.

Therapeutic compositions can contain a physiologically tolerable carriertogether with the cell composition, and optionally at least oneadditional bioactive agent as described herein, dissolved or dispersedtherein as an active ingredient. In some cases, the therapeuticcomposition is not substantially immunogenic when administered to amammal or human patient for therapeutic purposes, unless so desired.

In general, the progenitor cells described herein can be administered asa suspension with a pharmaceutically acceptable carrier. One of skill inthe art can recognize that a pharmaceutically acceptable carrier to beused in a cell composition can not include buffers, compounds,cryopreservation agents, preservatives, or other agents in amounts thatsubstantially interfere with the viability of the cells to be deliveredto the subject. A formulation comprising cells can include e.g., osmoticbuffers that permit cell membrane integrity to be maintained, andoptionally, nutrients to maintain cell viability or enhance engraftmentupon administration. Such formulations and suspensions are known tothose of skill in the art and/or can be adapted for use with theprogenitor cells, as described herein, using routine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

Administration & Efficacy

The terms “administering,” “introducing” and “transplanting” are usedinterchangeably in the context of the placement of cells, e.g.,progenitor cells, into a subject, by a method or route that results inat least partial localization of the introduced cells at a desired site,such as a site of injury or repair, such that a desired effect(s) isproduced. The cells e.g., progenitor cells, or their differentiatedprogeny, can be administered by any appropriate route that results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, or even the life time of the patient, i.e.,long-term engraftment. For example, in some aspects described herein, aneffective amount of myogenic progenitor cells is administered via asystemic route of administration, such as an intraperitoneal orintravenous route.

The terms “individual”, “subject,” “host” and “patient” are usedinterchangeably herein and refer to any subject for whom diagnosis,treatment or therapy is desired. In some aspects, the subject is amammal. In some aspects, the subject is a human being.

When provided prophylactically, progenitor cells described herein can beadministered to a subject in advance of any symptom of DMD, e.g., priorto the development of muscle wasting. Accordingly, the prophylacticadministration of a muscle progenitor cell population can serve toprevent DMD.

When provided therapeutically, muscle progenitor cells can be providedat (or after) the onset of a symptom or indication of DMD, e.g., uponthe onset of muscle wasting.

The muscle progenitor cell population being administered according tothe methods described herein can comprise allogeneic muscle progenitorcells obtained from one or more donors. “Allogeneic” refers to a muscleprogenitor cell or biological samples comprising muscle progenitor cellsobtained from one or more different donors of the same species, wherethe genes at one or more loci are not identical. For example, a muscleprogenitor cell population being administered to a subject can bederived from one more unrelated donor subjects, or from one or morenon-identical siblings. In some cases, syngeneic muscle progenitor cellpopulations can be used, such as those obtained from geneticallyidentical animals, or from identical twins. The muscle progenitor cellscan be autologous cells; that is, the muscle progenitor cells areobtained or isolated from a subject and administered to the samesubject, i.e., the donor and recipient are the same.

The term “effective amount” refers to the amount of a population ofprogenitor cells or their progeny needed to prevent or alleviate atleast one or more signs or symptoms of DMD, and relates to a sufficientamount of a composition to provide the desired effect, e.g., to treat asubject having DMD. The term “therapeutically effective amount”therefore refers to an amount of progenitor cells or a compositioncomprising progenitor cells that is sufficient to promote a particulareffect when administered to a typical subject, such as one who has or isat risk for DMD. An effective amount would also include an amountsufficient to prevent or delay the development of a symptom of thedisease, alter the course of a symptom of the disease (for example butnot limited to, slow the progression of a symptom of the disease), orreverse a symptom of the disease. It is understood that for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using routine experimentation.

For use in the various aspects described herein, an effective amount ofprogenitor cells comprises at least 10² progenitor cells, at least 5×10²progenitor cells, at least 10³ progenitor cells, at least 5×10³progenitor cells, at least 10⁴ progenitor cells, at least 5×10⁴progenitor cells, at least 10⁵ progenitor cells, at least 2×10⁵progenitor cells, at least 3×10⁵ progenitor cells, at least 4×10⁵progenitor cells, at least 5×10⁵ progenitor cells, at least 6×10⁵progenitor cells, at least 7×10⁵ progenitor cells, at least 8×10⁵progenitor cells, at least 9×10⁵ progenitor cells, at least 1×10⁶progenitor cells, at least 2×10⁶ progenitor cells, at least 3×10⁶progenitor cells, at least 4×10⁶ progenitor cells, at least 5×10⁶progenitor cells, at least 6×10⁶ progenitor cells, at least 7×10⁶progenitor cells, at least 8×10⁶ progenitor cells, at least 9×10⁶progenitor cells, or multiples thereof. The progenitor cells can bederived from one or more donors, or can be obtained from an autologoussource. In some examples described herein, the progenitor cells can beexpanded in culture prior to administration to a subject in needthereof.

Modest and incremental increases in the levels of functional dystrophinexpressed in cells of patients having DMD can be beneficial forameliorating one or more symptoms of the disease, for increasinglong-term survival, and/or for reducing side effects associated withother treatments. Upon administration of such cells to human patients,the presence of muscle progenitors that are producing increased levelsof functional dystrophin is beneficial. In some cases, effectivetreatment of a subject gives rise to at least about 3%, 5%, or 7%functional dystrophin relative to total dystrophin in the treatedsubject. In some examples, functional dystrophin will be at least about10% of total dystrophin. In some examples, functional dystrophin will beat least about 20% to 30% of total dystrophin. Similarly, theintroduction of even relatively limited subpopulations of cells havingsignificantly elevated levels of functional dystrophin can be beneficialin various patients because in some situations normalized cells willhave a selective advantage relative to diseased cells. However, evenmodest levels of muscle progenitors with elevated levels of functionaldystrophin can be beneficial for ameliorating one or more aspects of DMDin patients. In some examples, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90% or more ofthe muscle progenitors in patients to whom such cells are administeredare producing increased levels of functional dystrophin.

“Administered” refers to the delivery of a progenitor cell compositioninto a subject by a method or route that results in at least partiallocalization of the cell composition at a desired site. A cellcomposition can be administered by any appropriate route that results ineffective treatment in the subject, i.e. administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least 1×10⁴ cells are delivered tothe desired site for a period of time. Modes of administration includeinjection, infusion, instillation, or ingestion. “Injection” includes,without limitation, intravenous, intramuscular, intra-arterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. In someexamples, the route is intravenous. For the delivery of cells,administration by injection or infusion can be made.

The cells are administered systemically. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” refer to theadministration of a population of progenitor cells other than directlyinto a target site, tissue, or organ, such that it enters, instead, thesubject's circulatory system and, thus, is subject to metabolism andother like processes.

The efficacy of a treatment comprising a composition for the treatmentof DMD can be determined by the skilled clinician. However, a treatmentis considered “effective treatment,” if any one or all of the signs orsymptoms of, as but one example, levels of functional dystrophin arealtered in a beneficial manner (e.g., increased by at least 10%), orother clinically accepted symptoms or markers of disease are improved orameliorated. Efficacy can also be measured by failure of an individualto worsen as assessed by hospitalization or need for medicalinterventions (e.g., reduced muscle wasting, or progression of thedisease is halted or at least slowed). Methods of measuring theseindicators are known to those of skill in the art and/or describedherein. Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human, or a mammal)and includes: (1) inhibiting the disease, e.g., arresting, or slowingthe progression of symptoms; or (2) relieving the disease, e.g., causingregression of symptoms; and (3) preventing or reducing the likelihood ofthe development of symptoms.

The treatment according to the present disclosure can ameliorate one ormore symptoms associated with DMD by increasing the amount of functionaldystrophin in the individual. Early signs typically associated with DMD,include for example, delayed walking, enlarged calf muscle (due to scartissue), and falling frequently. As the disease progresses, childrenbecome wheel chair bound due to muscle wasting and pain. The diseasebecomes life threatening due to heart and/or respiratory complications.

Kits

The present disclosure provides kits for carrying out the methodsdescribed herein. A kit can include one or more of a genome-targetingnucleic acid, a polynucleotide encoding a genome-targeting nucleic acid,a site-directed polypeptide, a polynucleotide encoding a site-directedpolypeptide, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the aspects of the methods described herein, or anycombination thereof.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a genome-targeting nucleic acid, and (2) the site directedpolypeptide or a vector comprising a nucleotide sequence encoding thesite-directed polypeptide, and (3) a reagent for reconstitution and/ordilution of the vector(s) and or polypeptide.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a genome-targeting nucleic acid, and (ii) a nucleotide sequenceencoding the site-directed polypeptide and (2) a reagent forreconstitution and/or dilution of the vector.

In some of the kits, the kit can comprise a single-molecule guidegenome-targeting nucleic acid. In any of the above kits, the kit cancomprise a double-molecule genome-targeting nucleic acid. In any of thekits, the kit can comprise two or more double-molecule guides orsingle-molecule guides. The kits can comprise a vector that encodes thenucleic acid targeting nucleic acid.

In any of the kits, the kit can further comprise a polynucleotide to beinserted to effect the desired genetic modification.

Components of a kit can be in separate containers, or combined in asingle container.

Any kit can further comprise one or more additional reagents, where suchadditional reagents are selected from a buffer, a buffer for introducinga polypeptide or polynucleotide into a cell, a wash buffer, a controlreagent, a control vector, a control RNA polynucleotide, a reagent forin vitro production of the polypeptide from DNA, adaptors for sequencingand the like. A buffer can be a stabilization buffer, a reconstitutingbuffer, a diluting buffer, or the like. A kit can also comprise one ormore components that can be used to facilitate or enhance the on-targetbinding or the cleavage of DNA by the endonuclease, or improve thespecificity of targeting.

In addition to the above-mentioned components, a kit can furthercomprise instructions for using the components of the kit to practicethe methods. The instructions for practicing the methods can be recordedon a suitable recording medium. For example, the instructions can beprinted on a substrate, such as paper or plastic, etc. The instructionscan be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or subpackaging), etc. The instructions can be present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g. CD-ROM, diskette, flash drive, etc. In someinstances, the actual instructions are not present in the kit, but meansfor obtaining the instructions from a remote source (e.g. via theInternet), can be provided. An example of this case is a kit thatcomprises a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions can be recorded on a suitablesubstrate.

Guide RNA Formulation

Guide RNAs of the present disclosure can be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. Guide RNA compositions can beformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In some cases, the pHcan be adjusted to a range from about pH 5.0 to about pH 8. In somecases, the compositions can comprise a therapeutically effective amountof at least one compound as described herein, together with one or morepharmaceutically acceptable excipients. Optionally, the compositions cancomprise a combination of the compounds described herein, or can includea second active ingredient useful in the treatment or prevention ofbacterial growth (for example and without limitation, anti-bacterial oranti-microbial agents), or can include a combination of reagents of thepresent disclosure.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients can include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Other Possible Therapeutic Approaches

Gene editing can be conducted using nucleases engineered to targetspecific sequences. To date there are four major types of nucleases:meganucleases and their derivatives, zinc finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs), andCRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficultyof design, targeting density and mode of action, particularly as thespecificity of ZFNs and TALENs is through protein-DNA interactions,while RNA-DNA interactions primarily guide Cas9. Cas9 cleavage alsorequires an adjacent motif, the PAM, which differs between differentCRISPR systems. Cas9 from Streptococcus pyogenes cleaves using a NGGPAM, CRISPR from Neisseria meningitidis can cleave at sites with PAMsincluding NNNNGATT, NNNNNGTTT and NNNNGCTT. A number of other Cas9orthologs target protospacer adjacent to alternative PAMs.

CRISPR endonucleases, such as Cas9, can be used in the methods of thepresent disclosure. However, the teachings described herein, such astherapeutic target sites, could be applied to other forms ofendonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or usingcombinations of nucleases. However, in order to apply the teachings ofthe present disclosure to such endonucleases, one would need to, amongother things, engineer proteins directed to the specific target sites.

Additional binding domains can be fused to the Cas9 protein to increasespecificity. The target sites of these constructs would map to theidentified gRNA specified site, but would require additional bindingmotifs, such as for a zinc finger domain. In the case of Mega-TAL, ameganuclease can be fused to a TALE DNA-binding domain. The meganucleasedomain can increase specificity and provide the cleavage. Similarly,inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain andrequire the sgRNA/Cas9 target site and adjacent binding site for thefused DNA-binding domain. This likely would require some proteinengineering of the dCas9, in addition to the catalytic inactivation, todecrease binding without the additional binding site

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease Fokl. Because Fokl functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active Fokl dimer to form. Upondimerization of the Fokl domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers, although considerable expertise isrequired to do this well. In most applications of ZFNs, proteins of 4-6fingers are used, recognizing 12-18 bp respectively. Hence, a pair ofZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the Fokl domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese Fokl variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999);Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J BiolChem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97(2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).

Transcription Activator-Like Effector Nucleases (TALENs)

TALENs represent another format of modular nucleases whereby, as withZFNs, an engineered DNA binding domain is linked to the Fokl nucleasedomain, and a pair of TALENs operate in tandem to achieve targeted DNAcleavage. The major difference from ZFNs is the nature of the DNAbinding domain and the associated target DNA sequence recognitionproperties. The TALEN DNA binding domain derives from TALE proteins,which were originally described in the plant bacterial pathogenXanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acidrepeats, with each repeat recognizing a single basepair in the targetDNA sequence that is typically up to 20 bp in length, giving a totaltarget sequence length of up to 40 bp. Nucleotide specificity of eachrepeat is determined by the repeat variable diresidue (RVD), whichincludes just two amino acids at positions 12 and 13. The bases guanine,adenine, cytosine and thymine are predominantly recognized by the fourRVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. Thisconstitutes a much simpler recognition code than for zinc fingers, andthus represents an advantage over the latter for nuclease design.Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs arenot absolute in their specificity, and TALENs have also benefitted fromthe use of obligate heterodimer variants of the Fokl domain to reduceoff-target activity.

Additional variants of the Fokl domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive Fokl domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9/Cpf1 “nickase”mutants in which one of the Cas9 cleavage domains has been deactivated.DNA nicks can be used to drive genome editing by HDR, but at lowerefficiency than with a DSB. The main benefit is that off-target nicksare quickly and accurately repaired, unlike the DSB, which is prone toNHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012);and Moscou et al., Science 326(5959):1501 (2009). The use of TALENsbased on the “Golden Gate” platform, or cloning scheme, has beendescribed by multiple groups; see, e.g., Cermak et al., Nucleic AcidsRes. 39(12):e82 (2011); Li et al., Nucleic Acids Res.39(14):6315-25(2011); Weber et al., PLoS One. 6(2):e16765 (2011); Wanget al., J Genet Genomics 41(6):339-47, Epub 2014 May 17 (2014); andCermak T et al., Methods Mol Biol. 1239:133-59 (2015).

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingLAGLIDADG (SEQ ID NO. 1,410,474), GIY-YIG, His-Cis box, H-N-H,PD-(D/E)xK, and Vsr-like that are derived from a broad range of hosts,including eukarya, protists, bacteria, archaea, cyanobacteria and phage.As with ZFNs and TALENs, HEs can be used to create a DSB at a targetlocus as the initial step in genome editing. In addition, some naturaland engineered HEs cut only a single strand of DNA, thereby functioningas site-specific nickases. The large target sequence of HEs and thespecificity that they offer have made them attractive candidates tocreate site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology 24(8):663-80 (2014); Belfort andBonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome55(8):553-69 (2012); and references cited therein.

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601(2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel andScharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-Tevl (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29(2014). It is anticipated that other combinations of existingnuclease-based approaches will evolve and be useful in achieving thetargeted genome modifications described herein.

dCas9-Fokl or dCpf1-Fok1 and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpf1 catalytic function—retaining onlythe RNA-guided DNA binding function—and instead fusing a Fokl domain tothe deactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76(2014); and Guilinger et al., Nature Biotech. 32: 577-82 (2014). BecauseFokl must dimerize to become catalytically active, two guide RNAs arerequired to tether two Fokl fusions in close proximity to form the dimerand cleave DNA. This essentially doubles the number of bases in thecombined target sites, thereby increasing the stringency of targeting byCRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-Tevl, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-Tevl, with the expectation that off-targetcleavage can be further reduced.

Methods and Compositions of the Invention

Accordingly, the present disclosure relates in particular to thefollowing non-limiting inventions: In a first method, Method 1, thepresent disclosure provides a method for editing a dystrophin gene in ahuman cell by genome editing, the method comprising the step of:introducing into the human cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the dystrophin gene thatresults in a permanent deletion, insertion, or replacement of one ormore exons or aberrant intronic splice acceptor or donor sites within ornear the dystrophin gene and results in restoration of the dystrophinreading frame and restoration of the dystrophin protein activity.

In another method, Method 2, the present disclosure provides a methodfor editing a dystrophin gene in a human cell by genome editing, asprovided in Method 1, wherein the human cell is a muscle cell or muscleprecursor cell.

In another method, Method 3, the present disclosure provides an ex vivomethod for treating a patient with Duchenne Muscular Dystrophy (DMD),the method comprising the steps of: i) creating a DMD patient specificinduced pluripotent stem cell (iPSC); ii) editing within or near adystrophin gene of the iPSC; iii) differentiating the genome-edited iPSCinto a Pax7+ muscle progenitor cell; and iv) implanting the Pax7+ muscleprogenitor cell into the patient.

In another method, Method 4, the present disclosure provides an ex vivomethod for treating a patient with DMD, as provided in Method 3, whereinthe creating step comprises: a) isolating a somatic cell from thepatient; and b) introducing a set of pluripotency-associated genes intothe somatic cell to induce the somatic cell to become a pluripotent stemcell.

In another method, Method 5, the present disclosure provides an ex vivomethod for treating a patient with DMD, as provided in Method 4, whereinthe somatic cell is a fibroblast.

In another method, Method 6, the present disclosure provides an ex vivomethod for treating a patient with DMD, as provided in Methods 4 and 5,wherein the set of pluripotency-associated genes is one or more of thegenes selected from the group consisting of OCT4, SOX2, KLF4, Lin28,NANOG and cMYC.

In another method, Method 7, the present disclosure provides an ex vivomethod for treating a patient with DMD, as provided in any one ofMethods 3-6, wherein the editing step comprises introducing into theiPSC one or more deoxyribonucleic acid (DNA) endonucleases to effect oneor more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the dystrophin gene that results in a permanent deletion,insertion, or replacement of one or more exons or aberrant intronicsplice acceptor or donor sites within or near the dystrophin gene andresults in restoration of the dystrophin reading frame and restorationof the dystrophin protein activity.

In another method, Method 8, the present disclosure provides an ex vivomethod for treating a patient with DMD, as provided in any one ofMethods 3-7, wherein the differentiating step comprises one or more ofthe following to differentiate the genome-edited iPSC into a Pax7+muscle progenitor cell: contacting the genome-edited iPSC with specificmedia formulations, including small molecule drugs; transgeneoverexpression; or serum withdrawal.

In another method, Method 9, the present disclosure provides an ex vivomethod for treating a patient with DMD, as provided in any one ofMethods 3-8, wherein the implanting step comprises implanting the Pax7+muscle progenitor cell into the patient by local injection into thedesired muscle.

In another method, Method 10, the present disclosure provides an in vivomethod for treating a patient with DMD, the method comprising the stepof editing a dystrophin gene in a cell of the patient.

In another method, Method 11, the present disclosure provides an in vivomethod for treating a patient with DMD, as provided in Method 10,wherein the editing step comprises introducing into the cell of thepatient one or more deoxyribonucleic acid (DNA) endonucleases to effectone or more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the dystrophin gene that results in a permanent deletion,insertion, or replacement of one or more exons or aberrant intronicsplice acceptor or donor sites within or near the dystrophin gene andresults in restoration of the dystrophin reading frame and restorationof the dystrophin protein activity.

In another method, Method 12, the present disclosure provides an in vivomethod for treating a patient with DMD, as provided in Method 11,wherein the cell is a muscle cell or muscle precursor cell.

In another method, Method 13, the present disclosure provides an in vivomethod for treating a patient with DMD, as provided in any one ofMethods 1, 7, and 11, wherein the one or more DNA endonucleases is aCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, arecombination of the naturally occurring molecule thereof, acodon-optimized thereof, modified version thereof, and combinationsthereof.

In another method, Method 14, the present disclosure provides a methodas provided in Method 13, wherein the method comprises introducing intothe cell one or more polynucleotides encoding the one or more DNAendonucleases.

In another method, Method 15, the present disclosure provides a methodas provided in Method 13, wherein the method comprises introducing intothe cell one or more ribonucleic acids (RNAs) encoding the one or moreDNA endonucleases.

In another method, Method 16, the present disclosure provides a methodas provided in Methods 14 and 15, wherein the one or morepolynucleotides or one or more RNAs is one or more modifiedpolynucleotides or one or more modified RNAs.

In another method, Method 17, the present disclosure provides a methodas provided in Method 13, wherein the one or more DNA endonuclease isone or more proteins or polypeptides.

In another method, Method 18, the present disclosure provides a methodas provided in any one of Methods 1-17, wherein the method furthercomprises introducing into the cell one or more guide ribonucleic acids(gRNAs).

In another method, Method 19, the present disclosure provides a methodas provided in Method 18, wherein the one or more gRNAs aresingle-molecule guide RNA (sgRNAs).

In another method, Method 20, the present disclosure provides a methodas provided in Methods 18 and 19, wherein the one or more gRNAs or oneor more sgRNAs is one or more modified gRNAs or one or more modifiedsgRNAs.

In another method, Method 21, the present disclosure provides a methodas provided in any one of Methods 18-20, wherein the one or more DNAendonucleases is pre-complexed with one or more gRNAs or one or moresgRNAs.

In another method, Method 22, the present disclosure provides a methodas provided in any one of Methods 1-21, wherein the method furthercomprises introducing into the cell a polynucleotide donor templatecomprising at least a portion of the wild-type dystrophin gene or cDNA.

In another method, Method 23, the present disclosure provides a methodas provided in Method 22, wherein the at least a portion of thewild-type dystrophin gene or cDNA includes at least a part of exon 1,exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10,exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18,exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26,exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34,exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42,exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50,exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58,exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66,exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74,exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions, syntheticintronic regions, fragments, combinations thereof, or the entiredystrophin gene or cDNA.

In another method, Method 24, the present disclosure provides a methodas provided in Method 22, wherein the at least a portion of thewild-type dystrophin gene or cDNA includes exon 1, exon 2, exon 3, exon4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12,exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20,exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28,exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36,exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44,exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52,exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60,exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68,exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76,exon 77, exon 78, exon 79, intronic regions, synthetic intronic regions,fragments, combinations thereof, or the entire dystrophin gene or cDNA.

In another method, Method 25, the present disclosure provides a methodas provided in any one of Methods 22-24, wherein the donor template is asingle or double stranded polynucleotide.

In another method, Method 26, the present disclosure provides a methodas provided in any one of Methods 1, 7, and 11, wherein the methodfurther comprises introducing into the cell one or more guideribonucleic acid (gRNAs), and wherein the one or more DNA endonucleasesis one or more Cas9 or Cpf1 endonucleases that effect a pair ofsingle-strand breaks (SSBs) or double-strand breaks (DSBs), the firstSSB or DSB break at a 5′ locus and the second SSB or DSB break at a 3′locus, that results in a permanent deletion or replacement of one ormore exons or aberrant intronic splice acceptor or donor sites betweenthe 5′ locus and the 3′ locus within or near the dystrophin gene andresults in restoration of the dystrophin reading frame and restorationof the dystrophin protein activity.

In another method, Method 27, the present disclosure provides a methodas provided in Method 26, wherein one gRNA creates a pair of SSBs orDSBs.

In another method, Method 28, the present disclosure provides a methodas provided in Method 26, wherein one gRNA comprises a spacer sequencethat is complementary to either the 5′ locus, the 3′ locus, or a segmentbetween the 5′ locus and 3′ locus.

In another method, Method 29, the present disclosure provides a methodas provided in Method 26, wherein the method comprises a first gRNA anda second gRNA, wherein the first gRNA comprises a spacer sequence thatis complementary to a segment of the 5′ locus and the second gRNAcomprises a spacer sequence that is complementary to a segment of the 3′locus.

In another method, Method 30, the present disclosure provides a methodas provided in Methods 26-29, wherein the one or more gRNAs are one ormore single-molecule guide RNAs (sgRNAs).

In another method, Method 31, the present disclosure provides a methodas provided in Methods 26-30, wherein the one or more gRNAs or one ormore sgRNAs are one or more modified gRNAs or one or more modifiedsgRNAs.

In another method, Method 32, the present disclosure provides a methodas provided in any one of Methods 26-31, wherein the one or more DNAendonucleases is pre-complexed with one or more gRNAs or one or moresgRNAs.

In another method, Method 33, the present disclosure provides a methodas provided in any one of Methods 26-32, wherein there is a deletion ofthe chromosomal DNA between the 5′ locus and the 3′ locus.

In another method, Method 34, the present disclosure provides a methodas provided in any one of Methods 26-33, wherein the deletion is asingle exon deletion.

In another method, Method 35, the present disclosure provides a methodas provided in Method 34, wherein the single exon deletion is a deletionof exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51,exon 52, or exon 53.

In another method, Method 36, the present disclosure provides a methodas provided in Methods 34 or 35, wherein the 5′ locus is proximal to a5′ boundary of a single exon selected from the group consisting of exon2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon52, and exon 53.

In another method, Method 37, the present disclosure provides a methodas provided in any one of Methods 34-36, wherein the 3′ locus isproximal to a 3′ boundary of a single exon selected from the groupconsisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon50, exon 51, exon 52, and exon 53.

In another method, Method 38, the present disclosure provides a methodas provided in any one of Methods 34-37, wherein the 5′ locus isproximal to a 5′ boundary and the 3′ locus is proximal to the 3′boundary of a single exon selected from the group consisting of exon 2,exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52,and exon 53.

In another method, Method 39, the present disclosure provides a methodas provided in any one of Methods 36-38, wherein proximal to theboundary of the exon includes the surrounding splice donors andacceptors of the neighboring intron.

In another method, Method 40, the present disclosure provides a methodas provided in any one of Methods 26-33, wherein the deletion is amulti-exon deletion.

In another method, Method 41, the present disclosure provides a methodas provided in Method 40, wherein the multi-exon deletion is a deletionof exons 45-53 or exons 45-55.

In another method, Method 42, the present disclosure provides a methodas provided in any one of Methods 40-41, wherein the 5′ locus isproximal to a 5′ boundary of multiple exons selected from the groupconsisting of exons 45-53 and exons 45-55.

In another method, Method 43, the present disclosure provides a methodas provided in any one of Methods 40-42, wherein the 3′ locus isproximal to a 3′ boundary of multiple exons selected from the groupconsisting of exons 45-53 and exons 45-55.

In another method, Method 44, the present disclosure provides a methodas provided in any one of Methods 40-43, wherein the 5′ locus isproximal to a 5′ boundary and a 3′ locus is proximal to the 3′ boundaryof multiple exons selected from the group consisting of exons 45-53 andexons 45-55.

In another method, Method 45, the present disclosure provides a methodas provided in any one of Methods 42-44, wherein proximal to theboundary of the exon includes the surrounding splice donors andacceptors of the neighboring intron.

In another method, Method 46, the present disclosure provides a methodas provided in any one of Methods 26-32, wherein there is a replacementof the chromosomal DNA between the 5′ locus and the 3′ locus.

In another method, Method 47, the present disclosure provides a methodas provided in any one of Methods 26-32 and 46, wherein the replacementis a single exon replacement.

In another method, Method 48, the present disclosure provides a methodas provided in any one of Methods 26-32 and 46-47, wherein the singleexon replacement is a replacement of exon 2, exon 8, exon 43, exon 44,exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70.

In another method, Method 49, the present disclosure provides a methodas provided in any one of Methods 47-48, wherein the 5′ locus isproximal to a 5′ boundary of a single exon selected from the groupconsisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon50, exon 51, exon 52, exon 53, or exon 70.

In another method, Method 50, the present disclosure provides a methodas provided in any one of Methods 47-49, wherein the 3′ locus isproximal to a 3′ boundary of a single exon selected from the groupconsisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon50, exon 51, exon 52, exon 53, or exon 70.

In another method, Method 51, the present disclosure provides a methodas provided in any one of Methods 47-50, wherein the 5′ locus isproximal to a 5′ boundary and a 3′ locus is proximal to the 3′ boundaryof a single exon selected from the group consisting of exon 2, exon 8,exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53,or exon 70.

In another method, Method 52, the present disclosure provides a methodas provided in any one of Methods 49-51, wherein proximal to theboundary of the exon includes the surrounding splice donors andacceptors of the neighboring intron or neighboring exon.

In another method, Method 53, the present disclosure provides a methodas provided in any one of Methods 26-32 or 46, wherein the replacementis a multi-exon replacement.

In another method, Method 54, the present disclosure provides a methodas provided in any one of Method 53, wherein the multi-exon replacementis a replacement of exons 45-53 or exons 45-55.

In another method, Method 55, the present disclosure provides a methodas provided in any one of Methods 53-54, wherein the 5′ locus isproximal to a 5′ boundary of multiple exons selected from the groupconsisting of exons 45-53 and exons 45-55.

In another method, Method 56, the present disclosure provides a methodas provided in any one of Methods 53-55, wherein the 3′ locus isproximal to a 3′ boundary of multiple exons selected from the groupconsisting of exons 45-53 and exons 45-55.

In another method, Method 57, the present disclosure provides a methodas provided in any one of Methods 53-56, wherein the 5′ locus isproximal to a 5′ boundary and a 3′ locus is proximal to the 3′ boundaryof multiple exons selected from the group consisting of exons 45-53 andexons 45-55.

In another method, Method 58, the present disclosure provides a methodas provided in any one of Methods 55-57, wherein proximal to theboundary of the exon includes the surrounding splice donors andacceptors of the neighboring intron.

In another method, Method 59, the present disclosure provides a methodas provided in any one of Methods 46-58, wherein the method furthercomprises introducing into the cell a polynucleotide donor templatecomprising at least a portion of the wild-type dystrophin gene or cDNA,and the replacement is by homology directed repair (HDR).

In another method, Method 60, the present disclosure provides a methodas provided in any one of Method 59, wherein the at least a portion ofthe wild-type dystrophin gene or cDNA includes at least a part of exon1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions,synthetic intronic regions, fragments, combinations thereof, or theentire dystrophin gene or cDNA.

In another method, Method 61, the present disclosure provides a methodas provided in any one of Method 59, wherein the at least a portion ofthe wild-type dystrophin gene or cDNA includes exon 1, exon 2, exon 3,exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon76, exon 77, exon 78, exon 79, intronic regions, synthetic intronicregions, fragments, combinations thereof, or the entire dystrophin geneor cDNA.

In another method, Method 62, the present disclosure provides a methodas provided in any one of Methods 1, 7, or 11, wherein the methodfurther comprises introducing into the cell one guide ribonucleic acid(gRNA) and a polynucleotide donor template comprising at least a portionof the wild-type dystrophin gene, and wherein the one or more DNAendonucleases is one or more Cas9 or Cpf1 endonucleases that effect onesingle-strand break (SSB) or double-strand break (DSB) at a locus withinor near the dystrophin gene that facilitates insertion of a new sequencefrom the polynucleotide donor template into the chromosomal DNA at thelocus that results in permanent insertion or correction of one or moreexons or aberrant intronic splice acceptor or donor sites within or nearthe dystrophin gene and results in restoration of the dystrophin readingframe and restoration of the dystrophin protein activity, and whereinthe gRNA comprises a spacer sequence that is complementary to a segmentof the locus.

In another method, Method 63, the present disclosure provides a methodas provided in any one of Methods 1, 7, or 11, wherein the methodfurther comprises introducing into the cell one or more guideribonucleic acid (gRNAs) and a polynucleotide donor template comprisingat least a portion of the wild-type dystrophin gene, and wherein the oneor more DNA endonucleases is one or more Cas9 or Cpf1 endonucleases thateffect a pair of single-strand breaks (SSBs) or double-strand breaks(DSBs), the first at a 5′ locus and the second at a 3′ locus, within ornear the dystrophin gene that facilitates insertion of a new sequencefrom the polynucleotide donor template into the chromosomal DNA betweenthe 5′ locus and the 3′ locus that results in a permanent insertion orcorrection of one or more exons or aberrant intronic splice acceptor ordonor sites between the 5′ locus and the 3′ locus within or near thedystrophin gene and results in restoration of the dystrophin readingframe and restoration of the dystrophin protein activity.

In another method, Method 64, the present disclosure provides a methodas provided in Method 63, wherein one gRNA creates a pair of SSBs orDSBs.

In another method, Method 65, the present disclosure provides a methodas provided in Method 63, wherein one gRNA comprises a spacer sequencethat is complementary to either the 5′ locus, the 3′ locus, or a segmentbetween the 5′ locus and the 3′ locus.

In another method, Method 66, the present disclosure provides a methodas provided in Method 63, wherein the method comprises a first gRNA anda second gRNA, wherein the first gRNA comprises a spacer sequence thatis complementary to a segment of the 5′ locus and the second gRNAcomprises a spacer sequence that is complementary to a segment of the 3′locus.

In another method, Method 67, the present disclosure provides a methodas provided in Methods 62 or 63, wherein the one or more gRNAs are oneor more single-molecule guide RNAs (sgRNAs).

In another method, Method 68, the present disclosure provides a methodas provided in Methods 62-63 or 67, wherein the one or more gRNAs or oneor more sgRNAs are one or more modified gRNAs or one or more modifiedsgRNAs.

In another method, Method 69, the present disclosure provides a methodas provided in any one of Methods 62-63 or 67-68, wherein the one ormore DNA endonucleases is pre-complexed with one or more gRNAs or one ormore sgRNAs.

In another method, Method 70, the present disclosure provides a methodas provided in any one of Methods 62-69, wherein the insertion is asingle exon insertion

In another method, Method 71, the present disclosure provides a methodas provided in Method 70, wherein the single exon insertion is aninsertion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon50, exon 51, exon 52, exon 53 or exon 70.

In another method, Method 72, the present disclosure provides a methodas provided in any one of Methods 70-71, wherein the locus, 5′ locus, or3′ locus is proximal to a boundary of a single exon selected from thegroup consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46,exon 50, exon 51, exon 52, exon 53 and exon 70.

In another method, Method 73, the present disclosure provides a methodas provided in Method 72, wherein proximal to the boundary of the exonincludes the surrounding splice donors and acceptors of the neighboringintron or neighboring exon.

In another method, Method 74, the present disclosure provides a methodas provided in any one of Methods 62-69, wherein the insertion is amulti-exon insertion.

In another method, Method 75, the present disclosure provides a methodas provided in Method 74, wherein the multi-exon insertion is aninsertion of exons 45-53 or exons 45-55.

In another method, Method 76, the present disclosure provides a methodas provided in any one of Methods 74-75, wherein the locus, 5′ locus, or3′ locus is proximal to a boundary of multiple-exons selected from thegroup consisting of exons 45-53 or exons 45-55.

In another method, Method 77, the present disclosure provides a methodas provided in Method 76, wherein proximal to the boundary of the exonincludes the surrounding splice donors and acceptors of the neighboringintron.

In another method, Method 78, the present disclosure provides a methodas provided in any one of Methods 62 or 63, wherein the at least aportion of the wild-type dystrophin gene or cDNA includes at least apart of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8,exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16,exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24,exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32,exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40,exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48,exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56,exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64,exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72,exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronicregions, synthetic intronic regions, fragments, combinations thereof, orthe entire dystrophin gene or cDNA.

In another method, Method 79, the present disclosure provides a methodas provided in any one of Methods 62 or 63, wherein the at least aportion of the wild-type dystrophin gene or cDNA includes exon 1, exon2, exon 3, exon 4, exon 5, exon 6,

exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14,exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22,exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30,exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38,exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46,exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54,exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62,exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70,exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78,exon 79, intronic regions, synthetic intronic regions, fragments,combinations thereof, or the entire dystrophin gene or cDNA.

In another method, Method 80, the present disclosure provides a methodas provided in any one of Methods 62-79, wherein the insertion is byhomology directed repair (HDR).

In another method, Method 81, the present disclosure provides a methodas provided in any one of Methods 62-80, wherein the donor template is asingle or double stranded polynucleotide.

In another method, Method 82, the present disclosure provides a methodas provided in any one of Methods 26-81, wherein the Cas9 or Cpf1 mRNA,gRNA, and donor template are each formulated into separate lipidnanoparticles or all co-formulated into a lipid nanoparticle.

In another method, Method 83, the present disclosure provides a methodas provided in any one of Methods 26-81, wherein the Cas9 or Cpf1 mRNAis formulated into a lipid nanoparticle, and both the gRNA and donortemplate are delivered to the cell by an adeno-associated virus (AAV)vector.

In another method, Method 84, the present disclosure provides a methodas provided in any one of Methods 26-81, wherein the Cas9 or Cpf1 mRNAis formulated into a lipid nanoparticle, and the gRNA is delivered tothe cell by electroporation and donor template is delivered to the cellby an adeno-associated virus (AAV) vector.

In another method, Method 85, the present disclosure provides a methodas provided in any one of Methods 1-84, wherein the dystrophin gene islocated on Chromosome X: 31,117,228-33,344,609 (Genome ReferenceConsortium—GRCh38/hg38).

In a first composition, Composition 1, the present disclosure providesone or more guide ribonucleic acids (gRNAs) for editing a dystrophingene in a cell from a patient with Duchenne Muscular Dystrophy (DMD),the one or more gRNAs comprising a spacer sequence selected from thegroup consisting of the nucleic acid sequences in SEQ ID Nos:1-1,410,472 of the Sequence Listing.

In another composition, Composition 2, the present disclosure providesthe one or more gRNAs of Composition 1, wherein the one or more gRNAsare one or more single-molecule guide RNAs (sgRNAs).

In another composition, Composition 3, the present disclosure providesthe one or more gRNAs or sgRNAs of Compositions 1 or 2, wherein the oneor more gRNAs or one or more sgRNAs is one or more modified gRNAs or oneor more modified sgRNAs.

Definitions

The term “comprising” or “comprises” is used in reference tocompositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The term “consisting essentially of” refers to those elements requiredfor a given aspect. The term permits the presence of additional elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that aspect of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the aspect.

The singular forms “a,” “an,” and “the” include plural references,unless the context clearly dictates otherwise.

Any numerical range recited in this specification describes allsub-ranges of the same numerical precision (i.e., having the same numberof specified digits) subsumed within the recited range. For example, arecited range of “1.0 to 10.0” describes all sub-ranges between (andincluding) the recited minimum value of 1.0 and the recited maximumvalue of 10.0, such as, for example, “2.4 to 7.6,” even if the range of“2.4 to 7.6” is not expressly recited in the text of the specification.Accordingly, the Applicant reserves the right to amend thisspecification, including the claims, to expressly recite any sub-rangeof the same numerical precision subsumed within the ranges expresslyrecited in this specification. All such ranges are inherently describedin this specification such that amending to expressly recite any suchsub-ranges will comply with written description, sufficiency ofdescription, and added matter requirements, including the requirementsunder 35 U.S.C. § 112(a) and Article 123(2) EPC. Also, unless expresslyspecified or otherwise required by context, all numerical parametersdescribed in this specification (such as those expressing values,ranges, amounts, percentages, and the like) may be read as if prefacedby the word “about,” even if the word “about” does not expressly appearbefore a number. Additionally, numerical parameters described in thisspecification should be construed in light of the number of reportedsignificant digits, numerical precision, and by applying ordinaryrounding techniques. It is also understood that numerical parametersdescribed in this specification will necessarily possess the inherentvariability characteristic of the underlying measurement techniques usedto determine the numerical value of the parameter.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples, which provide illustrative non-limiting aspects ofthe invention.

The examples describe the use of the CRISPR system as an illustrativegenome editing technique to create defined therapeutic genomicdeletions, insertions, or replacements, collectively termed “genomicmodifications” herein, in the dystrophin gene (DMD gene) that lead topermanent deletion or correction of problematic exons from the genomiclocus that restore the dystrophin reading frame and restore thedystrophin protein activity.

Single gRNAs spanning different regions of the DMD gene were selectedand tested for cutting efficiencies (Table 5). gRNAs were targeted toexons, introns, and the splice acceptors of multiple areas of interestin the DMD gene. The naming convention for all gRNAs discussed in theExamples is: #(corresponding to the gRNA)—NN (Cas protein: SP—S.pyogenes, SA—S. aureus, NM—N. meningitides, ST—S. thermophiles, TD—T.denticola, Cpf1)—NN ##(SA—Splice acceptor, E—Exon, I—Intron).

TABLE 5 SEQ ID gRNA Name gRNA sequence NO: 1-NM-SA51 AGTCTGAGTAGGAGCTAA1410400 AATATT 2-NM-SA44 CTTGATCCATATGCTTTTA 1410401 CCTGC 3-NM-SA52ATATTTGTTCTTACAGGCAA 1410402 CAAT 1-ST-SA53 CTGATTCTGAATTCTTTCAA 5344942-ST-SA53 TTTTCCTTTTATTCTACTTG 534495 3-ST-SA46 TTCTTTTGTTCTTCTAGCCT537307 4-ST-SA46 GTTCTTCTAGCCTGGAGAAA 537308 5-ST-SA50ATCTTCTAACTTCCTCTTTA 536097 6-ST-SA43 TGTTTTAAAATTTTTATATT 5413221-SA-SA51 TGAGTAGGAGCTAAAATATT 485512 2-SA-SA45 TTGGTATCTTACAGGAACTC490807 3-SA-SA53 TGATTCTGAATTCTTTCAAC 482860 4-SA-SA53TTTCCTTTTATTCTAGTTGA 482861 5-SA-SA46 TTCTTCTAGCCTGGAGAAAG 4898146-SA-SA43 GlTTTAAAATTTTTATATTA 499467 7-SA-SA55 TCTGAACATTTGGTCCTTTG481421 8-SA-SA55 AACATTTGGTCCTTTGCAGG 481420 1-Cpf1-SA51TGCAAAAACCCAAAATATTTTAG 1410403 2-Cpf1-SA51 GCAAAAACCCAAAATATTTTAGC1410404 3-Cpf1-SA51 CAAAAACCCAAAATATTTTAGCT 1410405 4-Cpf1-SA45CCGCTGCCCAATGCCATCCTGGA 1410406 5-Cpf1-SA45 TGTTTTGCCTTTTTGGTATCTTA1410407 6-Cpf1-SA45 GTTTTGCCTTTTTGGTATCTTAC 1410408 7-Cpf1-SA45TTTTGCCTTTTTGGTATCTTACA 1410409 8-Cpf1-SA45 GCCTTTTTGGTATCTTACAGGAA1410410 9-Cpf1-SA45 CCTTTTTGGTATCTTACAGGAAC 1410411 10-Cpf1-SA45TGGTATCTTACAGGAACTCCAGG 1410412 11-Cpf1-SA53 TTTTTCCTTTTATTCTAGTTGAA1410413 12-Cpf1-SA53 TCCTTTTATTCTAGTTGAAAGAA 1410414 13-Cpf1-SA53CCTTTTATTCTAGTTGAAAGAAT 1410415 14-Cpf1-SA44 TCAACAGATCTGTCAAATCGCCT1410416 15-Cpf1-SA44 TCTTGATCCATATGCTTTTACCT 1410417 16-Cpf1-SA44CTTGATCCATATGCTTTTACCTG 1410418 17-Cpf1-SA44 TTGATCCATATGCTTTACCTGC1410419 18-Cpf1-SA46 GTTCTTCrAGCCTGGAGAAAGAA 1410420 19-Cpf1-SA46TTCTTCTAGCCTGGAGAAAGAAG 1410421 2O-Cpf1-SA46 ATTCTTCTTTCTCCAGGCTAGAA1410422 21-Cpf1-SA46 TTCTTCTTTCTCCAGGCTAGAAG 1410423 22-Cpf1-SA43TTGTAGACTATCTTTTATATTCT 1410424 23-Cpf1-SA43 TACTGTTTTAAAATTTTTATATT1410425 24-Cpf1-SA43 ACTGTTTTAAAATTTTTATATTA 1410426 25-Cpf1-SA43CTGTTTTAAAATTTTTATATTAC 1410427 26-Cpf1-SA43 AAAATTTTTATATTACAGAATAT1410428 27-Cpf1-SA43 AAATTTTTATATTACAGAATATA 1410429 1-SP-SA51AAAATATTTTAGCTCCTACT 145442 2-SP-SA51 TGCAAAAACCCAAAATATTT 1454433-SP-SA45 TGGTATCTTACAGGAACTCC 186216 4-SP-SA45 TTGGTATCTTACAGGAACTC186217 5-SP-SA45 TGCCATCCTGGAGTTCCTGT 186218 6-SP-SA45TTGCCTTTTTGGTATCTTAC 186219 7-SP-SA45 TTTGCCTTTTTGGTATCTTA 1862208-SP-SA53 TGATTCTGAATTCTTTCAAC 125451 9-SP-SA53 TTTCCTTTTATTCTAGTTGA125452 10-SP-SA53 AATTCTTTCAACTAGAATAA 125453 11-SP-SA53ATTTATTTTTCCTTTTATTC 125455 12-SP-SA53 AlTCTTTCAACTAGAATAAA 12545413-SP-SA44 AGATCTGTCAAATCGCCTGC 237600 14-SP-SA44 CAGATCTGTCAAATCGCCTG237599 15-SP-SA44 GTCAAATCGCCTGCAGGTAA 237602 16-SP-SA44GATCCATATGCTTTTACCTG 237603 17-SP-SA44 ATCCATATGCTTTTACCTGC 23760118-SP-SA46 TTGTTCTTCTAGCCTGGAGA 178873 19-SP-SA46 AlTCTTTTGTTCTTCTAGCC178869 20-SP-SA46 TTCTTCTAGCCTGGAGAAAG 178875 21-SP-SA46TTCTTCTTTCTCCAGGCTAG 178870 22-SP-SA46 TCTTTTGTTCTTCTAGCCTG 17887123-SP-SA46 AAGATATTCTTTTGTTCTTC 178868 24-SP-SA46 TTATTCTTCTTTCTCCAGGC178872 25-SP-SA46 AATTTTATTCTTUTTTcrcc 178874 26-SP-SA46CAATTTTATTUTTCTTTCTC 178876 27-SP-SA52 AATCCTGCATTGTTGCCTGT 13621328-SP-SA52 TAAGGGATATTTGTTCTTAC 136214 29-SP-SA52 CTAAGGGATATTTGTTCTTA136215 30-SP-SA50 ATGCTTTTCTGTTAAAGAGG 155685 31-SP-SA50TGTATGCTTTTCTGTTAAAG 155687 32-SP-SA50 TCTTCTAACTTCCTCTTTAA 15568633-SP-SA50 ATGTGTATGCTTTTCTGTTA 155689 34-SP-SA50 TTTTCTGTTAAAGAGGAAGT155684 35-SP-SA50 GTGTATGCTTTTCTGTTAAA 155688 36-SP-SA43TTTTATATTACAGAATATAA 252291 37-SP-SA43 GTTTTAAAATTTTTATATTA 25229238-SP-SA55 CTGAACATTTGGTCCTTTGC 114755 39-SP-SA55 CATTTGGTCUTTTGCAGGGT114751 40-SP-SA55 CTCGCTCACTCACCCTGCAA 114753 41-SP-SA55TCTGAACATTTGGTCCTTTG 114756 42-SP-SA55 TGGTCCTTTGCAGGGTGAGT 11475043-SP-SA55 TCTCGCTCACTCACCCTGCA 114752 44-SP-SA55 TGAACATTTGGTCCTTTGCA114754 1-SP-E51 CCTACTCAGACTGTTACTC 1410430 2-SP-E51 ACTCTGGTGACACAACCTG1410431 3-SP-E51 ACACAACCTGTGGTTACTA 1410432 4-SP-E51ATGTTGGAGGTACCTGCTC 1410433 5-SP-E51 TGCTCTGGCAGATTTCAAC 14104346-SP-E51 GCTCTGGCAGATTTCAACC 1410435 7-SP-E51 GGCAGATTTCAACCGGGCT1410436 8-SP-E51 TTGGACAGAACTTACCGAC 1410437 9-SP-E51CATCTCGTTGATATCCTCA 1410438 10-SP-E51 GGTAAGTTCTGTCCAAGCC 141043911-SP-E51 GGTTGAAATCTGCCAGAGC 1410440 12-SP-E51 GCAGGTACCTCCAACATCA1410441 13-SP-E51 GGCATTTCTAGTTTGGAGA 1410442 14-SP-E51CAGTTTCCTTAGTAACCAC 1410443 15-SP-E51 CCAGAGTAACAGTCTGAGT 141044416-SP-E45 GGTATCTTACAGGAACTCC 1410445 17-SP-E45 TCTTACAGGAACTCCAGGA1410446 18-SP-E45 AGGAACTCCAGGATGGCAT 1410447 19-SP-E45GGAACTCCAGGATGGCATT 1410448 20-SP-E45 CCAGGATGGCATTGGGCAG 141044921-SP-E45 TCAGAACATTGAATGCAAC 1410450 22-SP-E45 AGAACATTGAATGCAACTG1410451 23-SP-E45 ACAGATGCCAGTATTCTAC 1410452 24-SP-E45ATTGGGAAGCCTGAATCTG 1410453 25-SP-E45 GGGAAGCCTGAATCTGCGG 141045426-SP-E45 AGCCTGAATCTGCGGTGGC 1410455 27-SP-E45 CTCCTGCCACCGCAGATTC1410456 28-SP-E45 CCGCTGCCCAATGCCATCC 1410457 29-SP-E53ACAAGAACACCTTCAGAAC 1410458 30-SP-E53 AGAACACCTTCAGAACCGG 141045931-SP-E53 GTTAAAGGATTCAACACAA 1410460 32-SP-E53 ACACAATGGCTGGAAGCTA1410461 33-SP-E53 GCTGAGCAGGTCTTAGGAC 1410462 34-SP-E53CAGAGCCAAGCTTGAGTCA 1410463 35-SP-E53 GCCAAGCTTGAGTCATGGA 141046436-SP-E53 ACAAGAACACCTTCAGAAC 1410465 37-SP-E53 AGAACACCTTCAGAACCGG1410466 38-SP-E53 GTTAAAGGATTCAACACAA 1410467 39-SP-E53ACACAATGGCTGGAAGCTA 1410468 40-SP-E53 AAGAAGCTGAGCAGGTCTT 141046941-SP-E53 GCTGAGCAGGTCTTAGGAC 1410470 42-SP-E53 CAGAGCCAAGCTTGAGTCA1410471 43-SP-E53 GCCAAGCTTGAGTCATGGA 1410472 1-SP-152ACAGTGGTTTAAGTAATCCG 136258 2-SP-I52 GGAGACATTCCGGAGTACCT 1362573-SP-I52 TTTGGAGAGCATCAGATTAC 136337 4-SP-I52 GTTTGGTGATTCTTACGGAC136306 5-SP-I52 TCTGTGTGACGTCAAAATTA 136275 6-SP-I52ATATGATGTTCTACCACATG 136406 1-SP-153 GCCCACCCTACTACGGCATA 1360932-SP-I53 CTGTACCTTATGCCGTAGTA 136089 3-SP-I53 ACTGTACCTTATGCCGTAGT136090 4-SP-I53 TACCTTATGCCGTAGTAGGG 136087 5-SP-I53ACCTTATGCCGTAGTAGGGT 136086 6-SP-I53 TGCACAGCGTCTAGTCAGAT 1360791-SP-144 CATCGCATAGTTTAGTATAT 237710 2-SP-I44 CTTAGGTAAACATACAGCCC237749 3-SP-I44 ACTCCTTTCAGTTGATGAAC 237661 4-SP-I44AlTTTAGATTGGAATACTGC 237724 5-SP-I44 GCCTCAGTCTCTTTTATGAC 2377406-SP-I44 CTGCCTGTTCATCAACTGAA 237664 1-SP-145 AATATTAGAGCACGGTGCTA237546 2-SP-I45 CTCTATACAAATGCCAACGC 237393 3-SP-I45CAGATAAACCAGCTCCGTCC 237535 4-SP-I45 AGGGAAGCATCGTAACAGCA 2375215-SP-I45 ACTTGCATGCACACCAGCGT 237394 6-SP-I45 AGAGTTTGCCTGGACGGAGC237533 7-SP-I45 TTAGTGATCGTGGATACGAG 186301 8-SP-I45TTTGGGTTTCTTAGTGATCG 186298 9-SP-I45 AAAAACTGGAGCTAACCGAG 18626310-SP-I45 CATTCAGATTTAAATACGGT 186375 H-SP-145 AAAACTGGAGCTAACCGAGA186262 12-SP-I45 TTTGTAAGCTTGTCAGCTAG 186274 1-SP-146CAACTGCAGCAGCACGCATT 186065 2-SP-I46 CCACCTATTATGTGGATGAT 1860303-SP-I46 ATATACTTGTGGCTAGTTAG 186135 4-SP-I46 CCCATCATCCACATAATAGG186025 5-SP-I46 CCATTAAACTTGTACCTCTT 186083 6-SP-I46CCACCCATCATCCACATAAT 186027 1-SP-154 GCTGGGGACCGTTATCTATT 1211562-SP-I54 GCACATTCACGTATTACTGC 121149 3-SP-I54 TTTAGTTGAACGCCAGTAGA121051 4-SP-I54 CACATTCACGTATTACTGCT 121150 5-SP-I54ACATTCACGTATTACTGCTG 121151 6-SP-I54 CGTGAATGTGCTAGTTTTAC 1211471-SP-155 TAGCTCCCTATTATATCACG 120796 2-SP-I55 GCCAAGTCCGTGAGTTTAGT120916 3-SP-I55 CCTATTATATCACGTGGTTC 120798 4-SP-I55CCTGAACCACGTGATATAAT 120794 5-SP-I55 CTGAACCACGTGATATAATA 1207936-SP-I55 TTCTCATTTGATACATCCCC 120802 49-SP-I50 CATTGGCTTTGATTTCCCTA145522 51-SP-I51 ACAGTTGCCTAAGAACTGGT 145360 53-SP-I55GCCTTCTTTATCCCCTATCG 91033 44-SP-E70 ACTGGCAGGTAGCCCATTCG 1356245-SP-E70 TTTGCGAAGCATCCCCGAAT 13563 46-SP-E70 TTTTGCGAAGCATCCCCGAA13564 47-SP-E70 CACTGGCAGGTAGCCCATTC 13561 48-SP-E70GCACTGGCAGGTAGCCCATT 13560 54-SP-E55 AGGATGCTACCCGTAAGGAA 11470955-SP-E55 CCTTACGGGTAGCATCCTGT 114716 56-SP-E55 AACAACTGCCAATGTCCTAC114717 57-SP-E55 ATTACTGCAACAGTTCCCCC 114738 58-SP-E55GCAACAGTTCCCCCTGGACC 114736 59-SP-E55 TTCTAGGAGCUTTTCCTTAC 11471060-SP-E55 AGGCTCCTAGAAGACTCCAA 114700 61-SP-E55 GGTAGCATCCTGTAGGACAT114719 62-SP-E55 ACCTGGAAAAGTTTCTTGCC 114728 63-SP-E55GCCAGGCAAGAAACTTTTCC 114730

All tested gRNAs can be used for an HDR/correction based editingapproach. Single gRNAs targeting the splice acceptors can be used toinduce exon skipping to restore the reading frame of the DMD gene.Selected pairs of gRNAs can be used to make deletions in the DMD genethat restore the reading frame. Selected pairs of gRNAs can be used tomake deletions that simulate patient mutations and can be used togenerate model DMD mutant lines.

Various Cas orthologs were evaluated for cutting. SP, NM, ST, SA, andCpf1 gRNAs were delivered as RNA, expressed from the U6 promoter inplasmids, or expressed from the U6 promoter in lentivirus. Thecorresponding Cas protein was either knocked into the cell line ofinterest and constitutively expressed, delivered as mRNA, or deliveredas protein. The activity of the gRNAs in all the above mentioned formatswere evaluated using TIDE analysis or next generation sequencing inHEK293T cells, K562 cells, or induced pluripotent stem cells (iPSCs).

Overall, it was determined that most gRNAs tested induced cutting.However, the amount of cutting was highly dependent on the Cas proteintested. It was found that, generally, SP Cas9 gRNAs induce the highestlevels of cutting with SA Cas9 gRNAs inducing the second highest levelof cutting. Generally, it is beneficial to select gRNAs for therapeuticapplication that have the highest cutting efficiency possible. However,for an iPSC based therapy, the cutting efficiency is not as important.iPSCs are highly proliferative and make it simple to isolate a clonalpopulation of cells with the desired edit, even when the editingefficiency is less than 10%.

Introduction of the defined therapeutic modifications described aboverepresents a novel therapeutic strategy for the potential ameliorationof DMD, as further described and illustrated herein.

Example 1—CRISPR/SPCas9 Target Sites for the Dystrophin Gene

Regions of the dystrophin gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNRG. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 1-467,030. gRNA 19 bp spacersequences corresponding to the PAM were identified, as shown in SEQ IDNOs: 1,410,430-1,410,472 of the Sequence Listing.

Example 2—CRISPR/SACas9 Target Sites for the Dystrophin Gene

Regions of the dystrophin gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 467,031-528,196 of the SequenceListing.

Example 3—CRISPR/STCas9 Target Sites for the Dystrophin Gene

Regions of the dystrophin gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 24 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 528,197-553,198 of the SequenceListing.

Example 4—CRISPR/TDCas9 Target Sites for the Dystrophin Gene

Regions of the dystrophin gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 24 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 553,199-563,911 of the SequenceListing.

Example 5—CRISPR/NMCas9 Target Sites for the Dystrophin Gene

Regions of the dystrophin gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNNNGHTT. gRNA 24 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 563,912-627,854 and1,410,400-1,410,402 of the Sequence Listing.

Example 6—CRISPR/Cpf1 Target Sites for the Dystrophin Gene

Regions of the dystrophin gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceYTN. gRNA 20-24 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 627,855-1,410,399 and1,410,403-1,410,429 of the Sequence Listing.

Example 7—Illustrative Genome Editing Strategies Targeting Exon 2

Several methods provide gRNA pairs that delete exon 2 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 2 and the other gRNAcutting at the 3′ end of exon 2.

Example 8—Illustrative Genome Editing Strategies Targeting Exon 8

Several methods provide gRNA pairs that delete exon 8 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 8 and the other gRNAcutting at the 3′ end of exon 8.

Example 9—Illustrative Genome Editing Strategies Targeting Exon 43

Several methods provide gRNA pairs that delete exon 43 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 43 and the other gRNAcutting at the 3′ end of exon 43.

Example 10—Illustrative Genome Editing Strategies Targeting Exon 44

Several methods provide gRNA pairs that delete exon 44 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 44 and the other gRNAcutting at the 3′ end of exon 44.

Example 11—Illustrative Genome Editing Strategies Targeting Exon 45

Several methods provide gRNA pairs that delete exon 45 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 45 and the other gRNAcutting at the 3′ end of exon 45.

Example 12—Illustrative Genome Editing Strategies Targeting Exon 46

Several methods provide gRNA pairs that delete exon 46 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 46 and the other gRNAcutting at the 3′ end of exon 46.

Example 13—Illustrative Genome Editing Strategies Targeting Exon 50

Several methods provide gRNA pairs that delete exon 50 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 50 and the other gRNAcutting at the 3′ end of exon 50.

Example 14—Illustrative Genome Editing Strategies Targeting Exon 51

Several methods provide gRNA pairs that delete exon 51 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 51 and the other gRNAcutting at the 3′ end of exon 51.

Example 15—Illustrative Genome Editing Strategies Targeting Exon 52

Several methods provide gRNA pairs that delete exon 52 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 52 and the other gRNAcutting at the 3′ end of exon 52.

Example 16—Illustrative Genome Editing Strategies Targeting Exon 53

Several methods provide gRNA pairs that delete exon 53 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 53 and the other gRNAcutting at the 3′ end of exon 53.

Example 17—Illustrative Genome Editing Strategies Targeting Exon 70

Several methods provide gRNA pairs that delete exon 70 by cutting thegene twice, one gRNA cutting at the 5′ end of exon 70 and the other gRNAcutting at the 3′ end of exon 70.

Example 18—Illustrative Genome Editing Strategies Targeting Exons 45-53

Several methods provide gRNA pairs that delete exons 45-53 by cuttingthe gene twice, one gRNA cutting at the 5′ end of exon 45 and the othergRNA cutting at the 3′ end of exon 53.

Example 19—Illustrative Genome Editing Strategies Targeting Exons 45-55

Several methods provide gRNA pairs that delete exons 45-55 by cuttingthe gene twice, one gRNA cutting at the 5′ end of exon 45 and the othergRNA cutting at the 3′ end of exon 55.

Example 20—Bioinformatics Analysis of the Guide Strands

Candidate guides were screened and selected in a multi-step process thatinvolved both theoretical binding and experimentally assessed activity.By way of illustration, candidate guides having sequences that match aparticular on-target site, such as a site within or near the dystrophingene, with adjacent PAM can be assessed for their potential to cleave atoff-target sites having similar sequences, using one or more of avariety of bioinformatics tools available for assessing off-targetbinding, as described and illustrated in more detail below, in order toassess the likelihood of effects at chromosomal positions other thanthose intended. Candidates predicted to have relatively lower potentialfor off-target activity can then be assessed experimentally to measuretheir on-target activity, and then off-target activities at varioussites. Preferred guides have sufficiently high on-target activity toachieve desired levels of gene editing at the selected locus, andrelatively lower off-target activity to reduce the likelihood ofalterations at other chromosomal loci. The ratio of on-target tooff-target activity is often referred to as the “specificity” of aguide.

For initial screening of predicted off-target activities, there are anumber of bioinformatics tools known and publicly available that can beused to predict the most likely off-target sites; and since binding totarget sites in the CRISPR/Cas9 nuclease system is driven byWatson-Crick base pairing between complementary sequences, the degree ofdissimilarity (and therefore reduced potential for off-target binding)is essentially related to primary sequence differences: mismatches andbulges, i.e. bases that are changed to a non-complementary base, andinsertions or deletions of bases in the potential off-target siterelative to the target site. An exemplary bioinformatics tool calledCOSMID (CRISPR Off-target Sites with Mismatches, Insertions andDeletions) (available on the web at crispr.bme.gatech.edu) compiles suchsimilarities. Other bioinformatics tools include, but are not limitedto, GUIDO, autoCOSMID, and CCtop.

Bioinformatics were used to minimize off-target cleavage in order toreduce the detrimental effects of mutations and chromosomalrearrangements. Studies on CRISPR/Cas9 systems suggested the possibilityof high off-target activity due to nonspecific hybridization of theguide strand to DNA sequences with base pair mismatches and/or bulges,particularly at positions distal from the PAM region. Therefore, it isimportant to have a bioinformatics tool that can identify potentialoff-target sites that have insertions and/or deletions between the RNAguide strand and genomic sequences, in addition to base-pair mismatches.The bioinformatics-based tool, COSMID (CRISPR Off-target Sites withMismatches, Insertions and Deletions) was therefore used to searchgenomes for potential CRISPR off-target sites (available on the web atcrispr.bme.gatech.edu). COSMID output ranked lists of the potentialoff-target sites based on the number and location of mismatches,allowing more informed choice of target sites, and avoiding the use ofsites with more likely off-target cleavage.

Additional bioinformatics pipelines were employed that weigh theestimated on- and/or off-target activity of gRNA targeting sites in aregion. Other features that can be used to predict activity includeinformation about the cell type in question, DNA accessibility,chromatin state, transcription factor binding sites, transcriptionfactor binding data, and other CHIP-seq data. Additional factors areweighed that predict editing efficiency, such as relative positions anddirections of pairs of gRNAs, local sequence features andmicro-homologies.

Example 21—Testing of Preferred Guides in Cells for On-Target Activity

The gRNAs predicted to have the lowest off-target activity will then betested for on-target activity in epithelial cells derived from a humanembryonic kidney, HEK 293 Ts, by transient transfection and evaluatedfor indel frequency using TIDE or next generation sequencing. TIDE is aweb tool to rapidly assess genome editing by CRISPR-Cas9 of a targetlocus determined by a guide RNA (gRNA or sgRNA). Based on thequantitative sequence trace data from two standard capillary sequencingreactions, the TIDE software quantifies the editing efficacy andidentifies the predominant types of insertions and deletions (indels) inthe DNA of a targeted cell pool. See Brinkman et al, Nucl. Acids Res.(2014) for a detailed explanation and examples. Next-generationsequencing (NGS), also known as high-throughput sequencing, is thecatch-all term used to describe a number of different modern sequencingtechnologies including: Illumina (Solexa) sequencing, Roche 454sequencing, Ion torrent: Proton/PGM sequencing, and SOLiD sequencing.These recent technologies allow one to sequence DNA and RNA much morequickly and cheaply than the previously used Sanger sequencing, and assuch have revolutionized the study of genomics and molecular biology.HEK 293 Ts are a good model system for gene correction in iPSCs becauseboth cell types are known to have loose chromatin structures.

Chromatin is organizing by coiling into discrete structures callednucleosomes. This coiling influences accessibility of the genomicmaterial to transcriptional machinery. Regions of the genome that areopen are termed euchromatin, while regions of tight coiling are calledheterochromatin. It is a well accepted paradigm that stem cells have agenerally loose chromatin conformation and as cells differentiate intomore specialized cell types, certain regions of the genome become closedforming heterochromatin (Sims, R. J. and D. Reinberg (2009). “Stemcells: Escaping fates with open states.” Nature 460(7257): 802-803).

Example 22—Testing in Relevant Model Cell Lines

Once all of the guide RNAs are evaluated individually and effectivegRNAs are identified, all permutations of pairs of gRNAs will be testedin relevant model cell lines for their ability to modify the DNAsequence of the dystrophin gene that would be predicted to restore thedystrophin reading frame. Myoblast and iPSC cell lines withmodifications similar or identical to those found in patient sampleswere generated. The cells are treated with the different individual andpairwise combinations of gRNAs and a donor DNA template, if and asapplicable. Samples can then be evaluated for restoration of dystrophinexpression using one or more biological methods known to those skilledin the art, for example, an enzyme-linked immunosorbent assay (ELISA)that specifically recognizes the C terminus of the dystrophin protein(note that truncated proteins do not contain an intact C terminus). Thepairs of gRNAs that restore dystrophin expression can then be furtherevaluated by an additional biologic technique, such as Western blot toconfirm expression of the appropriate size of dystrophin protein.

Example 23—Testing Different Approaches for HDR Gene Editing

After testing the gRNAs for both on-target activity and off-targetactivity, exon correction and knock-in strategies will be tested for HDRgene editing.

For the exon correction approach, donor DNA template will be provided asa short single-stranded oligonucleotide, a short double-strandedoligonucleotide (PAM sequence intact/PAM sequence mutated), a longsingle-stranded DNA molecule (PAM sequence intact/PAM sequence mutated)or a long double-stranded DNA molecule (PAM sequence intact/PAM sequencemutated). In addition, the donor DNA template will be delivered by AAV.

For the DNA knock-in approach, a single-stranded or double-stranded DNAhaving homologous arms to the Xp21.2 locus can include 40 nt or more ofa first target exon (the first coding exon) of the dystrophin gene, thecomplete coding DNA sequence (CDS) of the dystrophin gene and 3′UTR ofthe dystrophin gene, and at least 40 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to theXp21.2 locus can include 80 nt or more of a first target exon (the firstcoding exon) of the dystrophin gene, the complete coding DNA sequence(CDS) of the dystrophin gene and 3′UTR of the dystrophin gene, and atleast 80 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the Xp21.2 locus caninclude 100 nt or more of a first target exon (the first coding exon) ofthe dystrophin gene, the complete coding DNA sequence (CDS) of thedystrophin gene and 3′UTR of the dystrophin gene, and at least 100 nt ofthe following intron. The single-stranded or double-stranded DNA havinghomologous arms to the Xp21.2 locus can include 150 nt or more of thefirst target exon (the first coding exon) of the dystrophin gene, thecomplete coding DNA sequence (CDS) of the dystrophin gene and 3′UTR ofthe dystrophin gene, and at least 150 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to theXp21.2 locus can include 300 nt or more of the first target exon (thefirst coding exon) of the dystrophin gene, the complete coding DNAsequence (CDS) of the dystrophin gene and 3′UTR of the dystrophin gene,and at least 300 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the Xp21.2 locus caninclude 400 nt or more of the first target exon (the first coding exon)of the dystrophin gene, the complete CDS of the dystrophin gene and3′UTR of the dystrophin gene, and at least 400 nt of the followingintron. Alternatively, the DNA template will be delivered by AAV.

For the cDNA knock-in approach, a single-stranded or double-strandedcDNA can include 40 nt or more of a single exon target of the dystrophingene. The single-stranded or double-stranded cDNA can include 80 nt ormore of a single exon target of the dystrophin gene. The single-strandedor double-stranded cDNA can include 100 nt or more of a single exontarget of the dystrophin gene. The single-stranded or double-strandedcDNA can include 150 nt or more of a single exon target of thedystrophin gene. The single-stranded or double-stranded cDNA can include300 nt or more of a single exon target of the dystrophin gene. Thesingle-stranded or double-stranded cDNA can include 400 nt or more of asingle exon target of the dystrophin gene. Alternatively, the DNAtemplate will be delivered by AAV.

For the cDNA knock-in approach, a single-stranded or double-strandedcDNA can include 40 nt or more of a multiple exon target of thedystrophin gene. The single-stranded or double-stranded cDNA can include80 nt or more of a multiple exon target of the dystrophin gene. Thesingle-stranded or double-stranded cDNA can include 100 nt or more of amultiple exon target of the dystrophin gene. The single-stranded ordouble-stranded cDNA can include 150 nt or more of a multiple exontarget of the dystrophin gene. The single-stranded or double-strandedcDNA can include 300 nt or more of a multiple exon target of thedystrophin gene. The single-stranded or double-stranded cDNA can include400 nt or more of a multiple exon target of the dystrophin gene.Alternatively, the DNA template will be delivered by AAV.

Example 24—Re-Assessment of Lead CRISPR-Cas9/DNA Donor Combinations

After testing the different strategies for HDR gene editing, the leadCRISPR-Cas9/DNA donor combinations will be re-assessed intherapeutically relevant cells for efficiency of deletion,recombination, and off-target specificity. Cas9 mRNA or RNP will beformulated into lipid nanoparticles for delivery, sgRNAs will beformulated into nanoparticles or delivered as AAV, and donor DNA will beformulated into nanoparticles or delivered as AAV.

Example 25—In Vivo Testing in Relevant Animal Model

After the CRISPR-Cas9/DNA donor combinations have been re-assessed, thelead formulations will be tested in vivo in a therapeutically relevantmouse model.

Culture in human cells allows direct testing on the human target and thebackground human genome, as described above.

Preclinical efficacy and safety evaluations can be observed throughengraftment of modified mouse or human cells in a therapeuticallyrelevant mouse model. The modified cells can be observed in the monthsafter engraftment.

Example 26—Cutting Efficiency of S. pyogenes gRNAs Targeting Exons 45,51, 53, 55, and 70 in the DMD Gene

S. pyogenes (SP) gRNAs were tested that target Exons 45, 51, 53, 55, and70 in the DMD gene (FIGS. 3A-3B). Each of Exons 45, 51, 53, 55, and 70may be edited using an HDR/correction based approach.

The SP gRNAs were cloned into plasmids that co-express the SP Casprotein. The plasmids were transfected into HEK293T cells usinglipofectamine 2000. Cells were harvested 48 hours post transfection, thegenomic DNA was isolated, and cutting efficiency was evaluated usingTIDE analysis. Data was compiled from one experiment containing 3-4replicates (N=3 to 4). Data was plotted as mean and SEM.

The data from FIGS. 3A-3B indicate that most gRNAs cut with efficienciesgreater than 50% in HEK293T cells.

Example 27—Cutting Efficiency of gRNAs Targeting the Splice Acceptor ofExons 43, 44, 45, 46, 50, 51, 52, 53, and 55 in the DMD Gene

A viable option for treating DMD is to induce exon skipping to restorethe reading frame of the DMD gene. To induce exon skipping, the geneediting approach must remove the AG sequence just upstream of the exonthat is recognized by endogenous splicing machinery. When a single gRNAinduces a double stranded break, the cell will repair the break. Somefraction of the time, the endogenous repair machinery will make amistake and either insert or delete bases adjacent to the cut site. ThegRNAs that mutate the AG sequence are likely to induce exon skipping atthis site as the splicing machinery will no longer be able to recognizethis site as a splice acceptor site and will skip to the next spliceacceptor of the neighboring exon.

S. pyogenes (SP), S. aureus (SA), S. thermophiles (ST), N. Meningitidis(NM), and Cpf1 gRNAs were designed and tested that target the spliceacceptor of Exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 in the DMD gene(FIGS. 4A, 4B, and 4C).

SP gRNAs were designed to target the splice acceptor of nine exons inthe DMD gene. The gRNAs were ordered as split RNA gRNAs from IntegratedDNA Technologies (IDT). The split gRNAs were annealed to the tracRNA permanufacturer's instructions. The annealed split gRNAs were thentransfected into HEK293T cells that stably express the SP Cas9 proteincells using RNAiMax. Cells were harvested 48 hours post transfection,the genomic DNA was isolated, and cutting efficiency was evaluated usingTIDE analysis (FIG. 4A). Data was compiled between two independentexperiments each containing 3 replicates (N=2 to 6). Data was plotted asmean and SEM.

NM, ST, and SA gRNAs were designed to target the splice acceptor of nineexons. The gRNAs were cloned into plasmids that co-express the Casprotein of interest along with the corresponding gRNA. The plasmids weretransfected into HEK293T cells using lipofectamine 2000. Cells wereharvested 48 hours post transfection, the genomic DNA was isolated, andcutting efficiency was evaluated using TIDE analysis (FIG. 4B). Data wascompiled between 2-4 independent experiments each containing 3replicates (N=6 to 12). Data was plotted as mean and SEM.

Cpf1 gRNAs were designed to target the splice acceptor of nine exons inthe DMD gene. The gRNAs were cloned into plasmids that express the gRNA.HEK293T were co-transfected with the gRNA plasmid of interest and asecond plasmid expressing Cpf1 using lipofectamine 2000. Cells wereharvested 48 hours post transfection, the genomic DNA was isolated, andcutting efficiency was evaluated using TIDE analysis (FIG. 4C). Data wascompiled between two independent experiments each containing 3replicates (N=3 to 6). Data was plotted as mean and SEM.

Example 28—Cutting Efficiencies and Splice Acceptor Knock-OutEfficiences of gRNAs Targeting Exons 43, 44, 45, 46, 50, 51, 52, 53, and55 in the DMD Gene

Many of the splice acceptor targeting gRNAs cut efficiently at thedesired splice site. To evaluate if the gRNA effectively knocked out thedesired AG sequence, PCR amplicons around the cut site were submittedfor next generation sequencing. The indel percentage reads where thesplice acceptor site was removed were quantified (FIGS. 5A-B and FIG. 6). A number of promising gRNAs were identified including, but notlimited to: 8-SA-SA55, 3-SP-SA45, 31-SP-SA50, and 40-SP-SA55 that removethe splice acceptor site in a large proportion of the reads.

S. pyogenes gRNAs were designed to target the splice acceptor of nineexons in the DMD gene. The gRNAs were ordered as split RNA gRNAs fromIDT. The split gRNAs were annealed to the tracRNA per manufacturer'sinstructions. The annealed split gRNAs were transfected into HEK293Tcells that stably express the S. pyogenes Cas9 protein cells usingRNAiMax. Cells were harvested 48 hours post transfection, the genomicDNA was isolated, and the desired 100-250 bp PCR amplicons surroundingthe desired splice acceptors were submitted for next generationsequencing. Data was compiled between two independent experiments eachcontaining 3 replicates (N=6). Averages were presented as populationaverages (FIGS. 5A-B).

N. meningitides (NM), S. thermophiles (ST), and S. aureus (SA) gRNAswere designed to target the splice acceptor of nine exons. The gRNAswere cloned into plasmids that co-expresses the Cas protein of interestalong with the corresponding gRNA. The plasmids were transfected intoHEK293T cells using lipofectamine 2000. Cells were harvested 48 hourspost transfection, the genomic DNA was isolated, and the desired 100-250bp PCR amplicons surrounding the desired splice acceptors were submittedfor next generation sequencing. Data was compiled between twoindependent experiments each containing 3 replicates (N=6). Averageswere presented as population averages (FIG. 6 ).

Example 29—Cutting Efficiency of gRNAs Targeting the Regions SurroundingExons 44, 45, 52, and 54 of the DMD Gene

To effectively evaluate editing approaches, it is important to accesspatient cell lines for in-vitro testing. However, patient material maybe difficult to access and there can be large patient-to-patientvariation between samples. Therefore, it was important to create DMDmutant cell lines that mimic common patient mutations. This allows theresearcher to test the efficacy of a repair strategy in the samebackground to ensure that variations in editing efficiency are notpatient specific. To address this, a variety of gRNAs were designed thatcan be paired to create common deletions found in DMD patients (Δ52,Δ44, Δ45, and Δ54). The resulting cell lines can be corrected usingeither an HDR or exon skipping approach. It is important to note, thatthese gRNAs can be used for either the creation of the model line or anHDR based correction of mutations of interest.

The region (100 bp-1 kb upstream and downstream of the exon of interest)was screened using gRNA design software. The best 6 gRNAs based onfewest predicted off target effects were selected on each side of theexon of interest (such as Exons 44, 45, 52, and 54 of the DMD gene). ThegRNAs were first evaluated in HEK293 Ts for cutting efficiency (FIGS.7A-7B) and confirmed for cutting efficiency in iPSC (FIGS. 8A-8B).

Single S. pyogenes gRNAs around exons 44, 45, 52, and 54 were selected.The gRNAs were ordered as split gRNA from IDT. Split gRNAs were annealedto the tracer sequence using manufacturer's instructions.

The annealed gRNAs were transfected into HEK293T that stably express theSP Cas9 protein cells using RNAiMax. Cells were harvested 48 hours posttransfection, the genomic DNA was isolated, and cutting efficiency wasevaluated using TIDE analysis (FIGS. 7A-7B). Data was compiled from oneexperiment each containing 3 replicates (N=1 to 3). Data was plotted asmean and SEM.

The annealed gRNAs were also subsequently complexed with Cas9 protein toform a ribonucleoprotein complex (RNP). The RNPs were transfected intoiPSCs (DiPS 1016SevA) using RNAiMax. Cells were harvested 48 hours posttransfection, the genomic DNA was isolated, and cutting efficiency wasevaluated using TIDE analysis (FIG. 8A-8B). Data was compiled from oneexperiment containing 3 replicates (N=1 to 3). Data was plotted as meanand SEM.

Single S. pyogenes gRNAs around exons 44, 45, 52, and 54 were selected.The gRNAs were ordered as split gRNA from IDT. Split gRNAs were annealedto the tracer sequence using manufacturer's instructions. The gRNAs weretransfected into HEK293T that stably express the SP Cas9 protein cellsusing RNAiMax. The same gRNAs were also complexed with Cas9 protein toform a ribonucleoprotein complex (RNP). The RNPs were transfected intoiPSCs (DiPS 1016SevA) using RNAiMax. Cells were harvested 48 hours posttransfection, the genomic DNA was isolated, and cutting was evaluatedusing TIDE analysis. Data was compiled from multiple experiments. Onlyaverage values were plotted.

There was a high correlation between editing efficiency in HEK293T cellsand iPSCs with a Pearson correlation coefficient of 0.51 overall. Assuch, screening in HEK293T cells was considered to be a good surrogatefor our therapeutic cell line of interest—iPSCs (FIG. 9 ).

Example 30—Clonal Analysis of Clonal Deletion Events

gRNAs with cutting efficiencies over 20% in iPSCs were identified ineach intron of interest except for intron 55. Single gRNAs were selectedwith the best cutting efficiencies to make the desired deletions.

Two pairs of gRNAs were used to create clonal Δ52 cell lines(1-SP-I52+2-SP-153 and 2-SP-I52+2-SP-I53). Out of 261 total clonesscreened, 57 had the desired deletion as accessed by PCR analysis (FIGS.10A, 10C).

To confirm the presence of the expected Δ52 deletion, genomic DNA fromeach clone of interest was harvested. PCR primers flanking the deletionwere designed. Since the deletion was small (<900 bp), a single pair ofprimers could be used to detect the deletion. This would result in asmaller deletion band (˜500 bp) or a wild-type (WT) band (˜1000 bp). Arepresentative gel of the deletion (del) or wild type (WT) product isshown in FIG. 10A.

The deletion even was confirmed in seven clones by submitting thedeletion PCR product for Sanger sequencing (7/7 clones had the predicteddeletion event with small insertions and deletions (FIGS. 11A-B). Thedeletion bands from seven clones (PCRs generated in FIG. 8A) were gelprepped and submitted for Sanger sequencing. Two clones created usinggRNA 1-SP-I52 and 2-SP-I53 were sequenced and aligned to the predicteddeletion product (assuming that S. pyogenes Cas9 cuts 3BP from the 3′end of the gRNA (FIG. 11A). Five clones created using gRNA 2-SP-I52 and2-SP-I53 were sequenced and aligned to the predicted deletion product(assuming that S. pyogenes Cas9 cuts 3BP from the 3′ end of the gRNA(FIG. 11B).

Similarly, two pairs of gRNAs were used to create clonal Δ44 cell lines(2-SP-I44+3-SP-I45 and 2-SP-I44+4-SP-I45). Out of 256 total clonesscreened, 16 had the desired deletion as accessed by PCR analysis (FIGS.10B, 10C).

To confirm the presence of the expected Δ44 deletion, genomic DNA fromeach clone of interest was harvested. Since the Δ44 gRNAs produce alarger deletion compared to the Δ52 gRNAs, two pairs of PCR primers weredesigned to either detect a deletion band or WT band. The expecteddeletion band was ˜400 bp and the expected WT band was ˜500 bp. Arepresentative gel of the deletion (del), wild type product (WT+), and anegative sample amplified with the wild type primers (WT-) is shown inFIG. 10B.

Example 31—Lentiviral Screen

To identify a large spectrum of pairs of gRNAs able to induce Exon51skipping, we conducted a large scale lentiviral screen. Intron 51 andIntron 52 genomic sequence were submitted for analysis using a gRNAdesign software. The resulting list of gRNAs were narrowed to about 3000left and 3000 right gRNAs adjacent to the Exon 51 splice acceptor. Thelist was narrowed based on uniqueness of sequence (only gRNAs without aperfect match somewhere else in the genome were screened) and minimalpredicted off targets. A left gRNA paired with a right gRNA shouldinduce Exon 51 skipping. The 6000 gRNAs were cloned into a lentiviralvector that expressed each gRNA of interest from the U6 promoter andconfers puromycin resistance. K562 cells were transduced with the virusat an MOI 2 and selected with puromycin to obtain a population of cellsthat were expressing a gRNA of interest. These cells were thennucleofected with Cas9 mRNA to induce a transient period of cutting.After 7 days, the cells were pelleted and the genomic DNA was extracted.The genomic DNA was enriched for the region of interest around Exon 51using hybrid capture. The enriched DNA was submitted for next generationsequencing (FIGS. 19A-19VV).

Example 32—In Vitro Transcribed (IVT) gRNA Screen

To identify a large spectrum of pairs of gRNAs able to induce Exon 45skipping, an in vitro transcribed (IVT) gRNA screen was conducted.Intron 45 and Intron 46 genomic sequence was submitted for analysisusing a gRNA design software. The resulting list of gRNAs were narrowedto a list of about 100 left and about 100 right gRNAs based onuniqueness of sequence (only gRNAs without a perfect match somewhereelse in the genome were screened) and minimal predicted off targets.This set of gRNAs were in vitro transcribed, and transfected usingmessenger Max into HEK293T cells that stably express Cas9. Cells wereharvested 48 hours post transfection, the genomic DNA was isolated, andcutting efficiencey was evaluated using TIDE analysis. (FIGS. 12A-E). Itwas found that about 18% of the tested gRNAs induced cuttingefficiencies over 50%.

Example 33—Partial cDNA Knockin Between Exons 45-55 of the DMD Gene

Another approach for correcting the DMD gene is a partial cDNA knock-in.As proof of principle, a study was conducted to replace the region Exon45-55 of the DMD gene (which could treat up to 62% of DMD patients). ThecDNA for Exon 45-55 is 3.2 kb. Two solid phase synthesized gRNAs [one inExon 45 (SEQ ID NO. 1410449) and a second in Exon 55 (SEQ ID NO 114738)]from Trilink were used for cutting. The two trilink gRNAs were complexedwith Cas9 protein and nucleofected into iPSCs along with a plasmiddonor. The plasmid donor was designed to have a 3.2 kb cassette (samesize as the desired cDNA knock-in) that constitutively expressed GFPwith 1 kb homology arms on each side to induce integration into the Exon45 to 55 site. The cells were tracked over 23 days. All experimentalconditions were nucleofected with high efficiency (over 60%); however,only GFP expression from cells that received the donor and gRNAstabilized over time, indicating that HDR occurred in about 16 percentof cells (FIG. 13A). The genomic DNA from these samples was isolated andtested for site specific integration of the donor construct. Sampleswere amplified with primers specific to the WT allele or the desiredknock-in allele. As expected, both the WT and knock-in allele could bedetected (FIG. 13B).

Example 34—Internally Deleted Yet Functional Dystrophin Protein

As proof of concept, it was demonstrated that we can edit in iPSC,isolate a clonal population of edited cells, and differentiate thosecells down the myogenic lineage to produce an internally deleted yetfunctional dystrophin protein.

One attractive method for correcting the DMD gene is to create a 445-55deletion. This deletion maintains the DMD reading frame and can restoreexpression of dystrophin in about 62% of DMD patients. To create thedesired 445-55 deletion, two published SP gRNAs (CR6: SEQ ID NO:1,410,475 and CR36: SEQ ID NO: 91033) were cloned into plasmids thatalso express SP Cas9 T2A orange florescent protein (OFP).

gRNA gRNA sequence PAM CR6 GGGGCTCCACCCTCACGAGT GGG CR36GCCTTCTTTATCCCCTATCG AGG

These two plasmids were co transfected into iPSCs using Mirus LT1transfection reagent. Two days later the cells expressing Cas9 asindicated by OFP expression were isolated using florescence activatedcell sorting (FACS). The cells were seeded at a low density and allowedto grow for 7-10 days until single cell clones appeared in the dishes.The clones were picked manually under a microscope and transferred into96 well plates. Once the cells reached confluence, the samples werepassaged 1:2 and genomic DNA was isolated from the remaining cells.

To identify clones with the desired deletion, a three primer PCR assaywas designed (FIGS. 14A and 14B). The assay allows for detection of awild type (WT) and deletion band in the same PCR reaction. Using thisassay, we identified 26/100 clones that had the editing event (FIG.14C).

To further validate that the clones had the desired Δ45-55 deletion, 5clones were submitted for Sanger sequencing. All five were confirmed tohave the desired deletion event. One clone had an insertion, and one asingle base pair deletion. The other three clones contained perfectdeletion events (FIG. 15 ).

All five clones that were sequenced, were also submitted forkaryotyping, and found karyotypically normal. Furthermore, all clonesmaintained pluripotency and stained over 99% positive for both SSEA-4and TRA-160 (FIGS. 16A-B).

Four of the clones were then differentiated into mytotubes using thepublished Chal et. al protocol. [Chal et. al (2015) Differentiation ofPluripotent stem cells to muscle fiber to model Duchenne MuscularDystrophy. Nature Biotechnology] to induce expression of dystrophin.Samples were harvested for Western blot and immuno-histo-chemistry. Allfive edited clones induced expression of an internally deleteddystrophin protein (FIG. 17 ). The sizes were compared to proteinisolated from HEK293T cells transfected with control cDNA plasmids thatexpress either the WT dystrophin protein or the 445-55 dystrophinprotein. The differentiated cells phenotypically produced myotubes asdemonstrated by myosin heavy chain staining. Representative image ofdifferentiated Clone 56 is shown in FIG. 18 .

Note Regarding Illustrative Aspects

While the present disclosure provides descriptions of various specificaspects for the purpose of illustrating various aspects of the presentinvention and/or its potential applications, it is understood thatvariations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should beunderstood to be at least as broad as they are claimed, and not as morenarrowly defined by particular illustrative aspects provided herein.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing descriptions, definitions,statements, or other disclosure material expressly set forth in thisspecification. As such, and to the extent necessary, the expressdisclosure as set forth in this specification supersedes any conflictingmaterial incorporated by reference. Any material, or portion thereof,that is said to be incorporated by reference into this specification,but which conflicts with existing definitions, statements, or otherdisclosure material set forth herein, is only incorporated to the extentthat no conflict arises between that incorporated material and theexisting disclosure material. Applicants reserve the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

1.-85. (canceled)
 86. A single-molecule guide RNA (sgRNA) comprising inthe 5′ to 3′ direction, a spacer sequence, a minimum CRISPR repeatsequence and a tracrRNA sequence, wherein the spacer sequence comprisesan RNA sequence encoded by SEQ ID NO:
 1410450. 87. The gRNA of claim 86,wherein the spacer sequence comprises from 19-25 nucleotides.
 88. ThesgRNA of claim 86, wherein the sgRNA is modified.
 89. The sgRNA of claim86, wherein the sgRNA is complexed with a Cas9 protein, optionallywherein the Cas9 protein comprises a nuclear localization signal.
 90. Amethod for treating a patient with Duchenne Muscular Dystrophy,comprising administering a nucleic acid encoding the sgRNA of claim 86.91. A method of treating a patient with Duchenne Muscular Dystrophy witha nucleic acid composition, wherein the nucleic acid compositioncomprises the nucleic acid of claim 86 and a second nucleic acidencoding a Cas9 endonuclease, optionally wherein the Cas9 endonucleasecomprises a nuclear localization signal, and optionally wherein the Cas9endonuclease is a Streptococcus pyogenes Cas9 endonuclease.
 92. Themethod of claim 91, wherein the nucleic acid composition is delivered tothe cell by a viral vector.
 93. The method of claim 92, wherein theviral vector is an adeno-associated virus (AAV) vector.
 94. The methodof claim 93, wherein the AAV vector is an AAV9 vector.
 95. The method ofclaim 90, wherein treating the patient with Duchenne Muscular Dystrophycomprises editing a dystrophin gene in a cell of the patient.
 96. Themethod of claim 91, wherein treating the patient with Duchenne MuscularDystrophy comprises editing a dystrophin gene in a cell of the patient.97. The method of claim 95, wherein the cell is a muscle cell or amuscle precursor cell.